Conditioned cell culture medium compositions and methods of use

ABSTRACT

Novel products comprising conditioned cell culture medium compositions and methods of use are described. The conditioned cell medium compositions of the invention may be comprised of any known defined or undefined medium and may be conditioned using any eukaryotic cell type. Once the cell medium of the invention is conditioned, it may be used in any state. Physical embodiments of the conditioned medium include, but are not limited to, liquid or solid, frozen, lyophilized or dried into a powder. Additionally, the medium is formulated with a pharmaceutically acceptable carrier as a vehicle for internal administration, applied directly to a food item or product, or formulated with a salve or ointment for topical applications. Also, the medium may be further processed to concentrate or reduce one or more factors or components contained within the medium.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.13/417,017, filed Mar. 9, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/349,451, filed Jan. 6, 2009, now U.S. Pat. No.8,138,147; which is a divisional of U.S. patent application Ser. No.11/538,380 filed Oct. 3, 2006, now U.S. Pat. No. 8,361,485; which is adivisional of Ser. No. 09/979,813 filed on Jul. 19, 2002, now U.S. Pat.No. 7,118,746; which is a 371 U.S. National Phase Application ofPCT/US00/13016 filed May 12, 2000; which is a continuation-in-part ofU.S. patent application Ser. No. 09/313,538 filed May 14, 1999, now U.S.Pat. No. 6,372,494. The disclosures of each of the above-referencedapplications are hereby incorporated by reference in their entirety.

INTRODUCTION

The invention relates to compositions comprising cell culture mediumconditioned by cells grown in two-dimensional culture (i. e., amonolayer), or in three-dimensional culture. The cells used to conditionthe medium may be genetically modified to alter the concentration ofproteins found in the medium. The conditioned cell medium is processedfor uses which include wound applications, cosmetic additives, foodsupplements, animal feed supplements, culturing cells, pharmaceuticalapplications, as well as compositions and methods for stimulating hairgrowth. The invention also relates to compositions containingextracellular matrix proteins and/or other purified protein(s) derivedfrom the conditioned medium.

BACKGROUND OF THE INVENTION Conditioned Cell Media

Culture medium compositions typically include essential amino acids,salts, vitamins, minerals, trace metals, sugars, lipids and nucleosides.Cell culture medium attempts to supply the components necessary to meetthe nutritional needs required to grow cells in a controlled, artificialand in vitro environment. Nutrient formulations, pH, and osmolarity varyin accordance with parameters such as cell type, cell density, and theculture system employed. Many cell culture medium formulations aredocumented in the literature and a number of media are commerciallyavailable. Once the culture medium is incubated with cells, it is knownto those skilled in the art as “spent” or “conditioned medium”.Conditioned medium contains many of the original components of themedium, as well as a variety of cellular metabolites and secretedproteins, including, for example, biologically active growth factors,inflammatory mediators and other extracellular proteins. Cell linesgrown as a monolayer or on beads, as opposed to cells grown inthree-dimensions, lack the cell-cell and cell-matrix interactionscharacteristic of whole tissue in vivo. Consequently, such cells secretea variety of cellular metabolites although they do not necessarilysecrete these metabolites and secreted proteins at levels that approachphysiological levels. Conventional conditioned cell culture medium,medium cultured by cell-lines grown as a monolayer or on beads, isusually discarded or occasionally used in culture manipulations such asreducing cell densities.

Tissue Culture Systems

The majority of vertebrate cell cultures in vitro are grown asmonolayers on an artificial substrate bathed in culture medium. Thenature of the substrate on which the monolayers grow may be solid, suchas plastic, or semisolid gels, such as collagen or agar. Disposableplastics have become the preferred substrate used in modern-day tissueor cell culture.

A few researchers have explored the use of natural substrates related tobasement membrane components. Basement membranes comprise a mixture ofglycoproteins and proteoglycans that surround most cells in vivo. Forexample, Reid and Rojkund, 1979, In, Methods in Enzymology, Vol. 57,Cell Culture, Jakoby & Pasten, eds., New York, Acad. Press, pp. 263-278;Vlodaysky et al., 1980, Cell 19:607-617; Yang et al., 1979, Proc. Natl.Acad. Sci. USA 76:3401 have used collagen for culturing hepatocytes,epithelial cells and endothelial tissue. Growth of cells on floatingcollagen (Michalopoulos and Pitot, 1975, Fed. Proc. 34:826) andcellulose nitrate membranes (Savage and Bonney, 1978, Exp. Cell Res.114:307-315) have been used in attempts to promote terminaldifferentiation. However, prolonged cellular regeneration and theculture of such tissues in such systems have not heretofore beenachieved.

Cultures of mouse embryo fibroblasts have been used to enhance growth ofcells, particularly at low densities. This effect is thought to be duepartly to supplementation of the medium but may also be due toconditioning of the substrate by cell products. In these systems, feederlayers of fibroblasts are grown as confluent monolayers which make thesurface suitable for attachment of other cells. For example, the growthof glioma on confluent feeder layers of normal fetal intestine has beenreported (Lindsay, 1979, Nature 228:80).

While the growth of cells in two dimensions is a convenient method forpreparing, observing and studying cells in culture, allowing a high rateof cell proliferation, it lacks characteristic of whole tissue in vivo.In order to study such functional and morphological interactions, a fewinvestigators have explored the use of three-dimensional substrates suchas collagen gel (Douglas et al., 1980, In Vitro 16:306-312; Yang et al.,1979, Proc. Natl. Acad. Sci. 76:3401; Yang et al., 1980, Proc. Natl.Acad. Sci. 77:2088-2092; Yang et al., 1981, Cancer Res. 41:1021-1027);cellulose sponge, alone (Leighton et al., 1951, J. Natl. Cancer Inst.12:545-561) or collagen coated (Leighton et al., 1968, Cancer Res.28:286-296); a gelatin sponge, Gelfoam (Sorour et al., 1975, J.Neurosurg. 43:742-749).

In general, these three-dimensional substrates are inoculated with thecells to be cultured. Many of the cell types have been reported topenetrate the matrix and establish a “tissue-like” histology. Forexample, three-dimensional collagen gels have been utilized to culturebreast epithelium (Yang et al., 1981, Cancer Res. 41:1021-1027) andsympathetic neurons (Ebendal, 1976, Exp. Cell Res. 98:159-169).Additionally, various attempts have been made to regenerate tissue-likearchitecture from dispersed monolayer cultures. (Kruse and Miedema,1965, J. Cell Biol. 27:273) reported that perfused monolayers could growto more than ten cells deep and organoid structures can develop inmultilayered cultures if kept supplied with appropriate medium (see alsoSchneider et al., 1963, Exp. Cell. Res. 30:449-459; Bell et al., 1979,Proc. Natl. Acad. Sci. USA 76:1274-1279; Green, 1978, Science200:1385-1388). It has been reported that human epidermal keratinocytesmay form dematoglyphs (friction ridges if kept for several weeks withouttransfer; Folkman and Haudenschild (1980, Nature 288:551-556) reportedthe formation of capillary tubules in cultures of vascular endothelialcells cultured in the presence of endothelial growth factor and mediumconditioned by tumor cells; and Sirica et al. (1979, Proc. Natl. Acad.Sci. USA 76:283-287; 1980, Cancer Res. 40:3259-3267) maintainedhepatocytes in primary culture for about 10-13 days on nylon meshescoated with a thin layer of collagen. However, the long term culture andproliferation of cells in such systems has not been achieved.

The establishment of long term culture of tissues such as bone marrowhas been attempted. Overall the results were disappointing, in thatalthough a stromal cell layer containing different cell types is rapidlyformed, significant hematopoiesis could not be maintained for any realtime. (For review see Dexter et al., In Long Term Bone Marrow Culture,1984, Alan R. Liss, Inc., pp. 57-96).

A number of groups have attempted to grow skin and connective tissue invitro for transplantation in vivo. In one such system, a hydrated bovinecollagen lattice forms the substrate to which cells, such as fibroblastsare incorporated which results in the contraction of the lattice intotissue (Bell et al., U.S. Pat. No. 4,485,096). In another system, aporous cross-linked collagen sponge is used to culture fibroblast cells(Eisenberg, WO 91/16010). A scaffold composed of synthetic polymers hasalso been described to control cell growth and proliferation in vitro sothat once the fibroblasts begin to grow and attach to the matrix it istransplanted into the patient (Vacanti et al., U.S. Pat. Nos. 5,759,830;5,770,193; 5,736,372).

Synthetic matrices composed of biodegradable, biocompatible copolymersof polyesters and amino acids have also been designed as scaffolding forcell growth (U.S. Pat. Nos. 5,654,381; 5,709,854). Non-biodegradablescaffolds are likewise capable of supporting cell growth.Three-dimensional cell culture systems have also been designed which arecomposed of a stromal matrix which supports the growth of cells from anydesired tissue into an adult tissue (Naughton et al., U.S. Pat. Nos.4,721,096 and 5,032,508). Another approach involves slowly polymerizinghydrogels containing large numbers of the desired cell type which hardeninto a matrix once administered to a patient (U.S. Pat. No. 5,709,854).Extracellular matrix preparations have been designed which are composedof stromal cells which provide a three dimensional cell culture systemfor a desired cell type which may be injected into the patient forprecise placement of the biomaterial (Naughton et al., WO 96/39101).

Cellular Cytokines and Growth Factors

The secretion of extracellular proteins into conditioned cell media suchas growth factors, cytokines, and stress proteins opens newpossibilities in the preparation of products for use in a large varietyof areas including tissue repair, e.g., in the treatment of wounds andother tissue defects such as cosmetic defects as well as human andanimal feed supplements. For example, growth factors are known to playan important role in the wound healing process. In general, it isthought desirable in the treatment of wounds to enhance the supply ofgrowth factors by direct addition of these factors.

Cellular cytokines and growth factors are involved in a number ofcritical cellular processes including cell proliferation, adhesion,morphologic appearance, differentiation, migration, inflammatoryresponses, angiogenesis, and cell death. Studies have demonstrated thathypoxic stress and injury to cells induce responses including increasedlevels of mRNA and proteins corresponding to growth factors such as PDGF(platelet-derived growth factor), VEGF (vascular endothelial growthfactor), FGF (fibroblast growth factor), and IGF (insulin-like growthfactor) (Gonzalez-Rubio, M. et al., 1996, Kidney Int. 50(1):164-73;Abramovitch, R. et al., 1997, Int J Exp. Pathol. 78(2):57-70; Stein, I.et al., 1995, Mol Cell Biol. 15(10):5363-8; Yang, W. et al., 1997, FEBSLett. 403(2):139-42; West, N. R. et al., 1995, J. Neurosci. Res.40(5):647-59).

Growth factors, such as transforming growth factor-β, also known in theart as TGF-β, are induced by certain stress proteins during woundhealing. Two known stress proteins are GRP78 and HSP90. These proteinsstabilize cellular structures and render the cells resistant to adverseconditions. The TGF-β3 family of dimeric proteins includes TGF-β1,TGF-β2, and TGF-β3 and regulates the growth and differentiation of manycell types. Furthermore, this family of proteins exhibits a range ofbiological effects, stimulating the growth of some cell types (Noda etal., 1989, Endocrinology 124:2991-2995) and inhibiting the growth ofother cell types (Goey et al., 1989, J. Immunol. M3:877-880; Pietenpolet al., 1990, Proc. Natl. Acad. Sci. USA 87:3758-3762). TGF-β has alsobeen shown to increase the expression of extracellular matrix proteinsincluding collagen and fibronectin (Ignotz et al., 1986, J. Biol. Chem.261:4337-4345) and to accelerate the healing of wounds (Mustoe et al.,1987, Science 237:1333-1335).

Another such growth factor is PDGF. PDGF was originally found to be apotent mitogen for mesenchymal-derived cells (Ross R. et al., 1974,Proc. Natl. Acad. Sci. USA 71(4):1207-1210; Kohler N. et al., 1974, Exp.Cell Res. 87:297-301). Further studies have shown that PDGF increasesthe rate of cellularity and granulation in tissue formation. Woundstreated with PDGF have the appearance of an early stage inflammatoryresponse including an increase in neutrophils and macrophage cell typesat the wound site. These wounds also show enhanced fibroblast function(Pierce, G. F. et al., 1988, J. Exp. Med. 167:974-987). Both PDGF andTGF-f3 have been shown to increase collagen formation, DNA content, andprotein levels in animal studies (Grotendorst, G. R. et al., 1985, J.Clin. Invest. 76:2323-2329; Sporn, M. B. et al., 1983, Science (WashD.C.) 219:1329). PDGF has been shown to be effective in the treatment ofhuman wounds. In human wounds, PDGF-AA expression is increased withinpressure ulcers undergoing healing. The increase of PDGF-AA correspondsto an increase in activated fibroblasts, extracellular matrixdeposition, and active vascularization of the wound. Furthermore, suchan increase in PDGF-AA is not seen in chronic non-healing wounds(Principles of Tissue Engineering, R. Lanza et al. (eds.), pp. 133-141(R.G. Landes Co. TX 1997). A number of other growth factors having theability to induce angiogenesis and wound healing include VEGF, KGF andbasic FGF.

There are currently no simple effective methods or compositions forapplication containing the variety of cytokines, growth factors or otherregulatory proteins found in Applicants' conditioned media.

SUMMARY OF THE INVENTION

The Applicants of the present invention have discovered novelconditioned cell culture medium compositions. Additionally, theinvention comprises uses for these novel compositions. The inventionfurther comprises compositions containing particular protein productsderived from the conditioned cell media of the invention.

The conditioned cell medium compositions of the invention may becomprised of any known defined or undefined medium and may beconditioned using any eukaryotic cell type. The medium may beconditioned by stromal cells, parenchymal cells, mesenchymal stem cells,liver reserve cells, neural stem cells, pancreatic stem cells, and/orembryonic stem cells. A three-dimensional tissue construct is preferred.The cell type, whether in monolayer or in three-dimensions, will affectthe properties of the conditioned medium. For example, a mediumconditioned with astrocytes and neuronal cells will elaborate certaincharacteristic metabolites and proteins so that such a conditionedmedium is preferred for certain nerve repair applications. In apreferred embodiment, Applicants' medium is conditioned with athree-dimensional cell and tissue culture. In another preferredembodiment, the medium is conditioned with the stromal cells used in theproduction of TransCyte™ (Smith & Nephew PLC., United Kingdom). In ahighly preferred embodiment, cells of the three-dimensional tissueculture are stromal cells and the tissue culture construct isDermagraft® (Advanced Tissue Sciences, Inc., La Jolla Calif.) with orwithout the addition of specific parenchymal cells. Such conditionedcell medium provides a unique combination of factors and specifiedratios that are different than monolayer cultures and more closelyrepresent those found in vivo. The three-dimensional stromal culture mayfurther be cultured with parenchymal cells such as the cells of theskin, bone, liver, nerve, pancreas, etc., resulting in a conditionedmedium containing characteristic extracellular proteins and othermetabolites of that tissue type. Additionally, each cell type may alsobe genetically modified. The genetic modification may be used to alterthe concentration of one or more component in the medium such as, forexample, to upregulate a protein, to introduce a new protein, or toregulate ion concentration.

Once the cell medium of the invention is conditioned, it may be used inany state. Physical embodiments of the conditioned medium include, butare not limited to, liquid or solid, frozen, lyophilized or dried into apowder. Additionally, the medium may be formulated with apharmaceutically acceptable carrier as a vehicle for internaladministration, applied directly to a food item or product, formulatedwith a salve or ointment for topical applications, or, for example, madeinto or added to surgical glue to accelerate healing of suturesfollowing invasive procedures. Also, the medium may be further processedto concentrate or reduce one or more factors or components containedwithin the medium. For example, the conditioned medium may be enrichedwith a growth factor by using immunoaffinity chromatography.

In one embodiment, the conditioned medium of the invention is used inwound healing. Examples include, but are not limited to, applying theconditioned cell medium to the gauze of a bandage (adhesive ornon-adhesive) and used in topical applications to promote and/oraccelerate wound healing. Again, the conditioned medium may be processedto concentrate or reduce one or more components to enhance woundhealing. The compositions may be lyophilized/freeze-dried and added as awound filler or added to existing wound filling compositions toaccelerate wound healing. Alternatively, the medium may be added to ahydrogel composition and used as a film for topical wound treatments andanti-adhesion applications. The medium compositions of the invention maybe conditioned with cells which express gene products with improvedwound-healing properties; i.e., engineered cells which express geneproducts that have anti-scarring properties.

In another embodiment, the conditioned cell medium formulations of theinvention are used to correct congenital anomalies and acquired physicaldefects. Further, formulations in the form of injectables or hydrogelsmay be used to eliminate wrinkles, frown lines, scarring and to repairother skin conditions. In another embodiment, the conditioned cellmedium may also be added to eye shadow, pancake makeup, compacts orother cosmetics.

In yet another embodiment, the conditioned cell media formulations ofthe invention are used as food additives and dietary supplements. Theconditioned medium contains a multitude of nutrients including essentialamino acids, vitamins, and minerals. The conditioned cell media of theinvention may be concentrated and/or lyophilized, for example, and arepreferably administered in capsules or tablets for ingestion.Additionally, the compositions can also be added directly to food toenhance its nutritional content.

In a further embodiment, the compositions may be used as a supplement toanimal feed as it contains a variety of proteins vitamins, antibiotics,polysaccharides and other factors beneficial for raising cattle andother ruminant animals.

In yet another embodiment of the invention, the compositions of theinvention may be used to culture cells. The conditioned cell media ofthe invention contains factors useful in promoting cell attachment andgrowth. Further, the cell medium may be conditioned by cells which aregenetically engineered and which may, for example, contain increasedfibronective or collagen concentrations beneficial in promoting cellattachment to a scaffold or culture surface.

In an additional embodiment of the invention, the conditioned cellmedium compositions of the invention are used for pharmaceuticalapplications. The invention comprises cell media cultured withthree-dimensional tissue constructs, such that, growth factors and otherproteins are secreted into the medium at ratios closely resembling thosefound in vivo. As such, the conditioned media of the invention isbeneficial for a variety of pharmaceutical applications.

Lastly, the compositions of the invention may be formulated for topicalapplications for stimulating hair growth.

Definitions

The following terms used herein shall have the meanings indicated:

Adherent Layer: cells attached directly to the three-dimensional supportor connected indirectly by attachment to cells that are themselvesattached directly to the support.

Conditioned Medium: a formulation containing extracellular protein(s)and cellular metabolites, which has previously supported the growth ofany desired eukaryotic cell type, said cells having been cultured ineither two or three dimensions. Also called “Conditioned Cell Medium” or“Conditioned Cell and Tissue Culture Medium”.

Stromal Cells: fibroblasts with or without other cells and/or elementsfound in loose connective tissue, including but not limited to,endothelial cells, pericytes, macrophages, monocytes, plasma cells, mastcells, adipocytes, mesenchymal stem cells, liver reserve cells, neuralstem cells, pancreatic stem cells, chondrocytes, prechondrocytes, etc.

Tissue-Specific or Parenchymal Cells: the cells which form the essentialand distinctive tissue of an organ as distinguished from its supportiveframework.

Three-Dimensional Framework: a three-dimensional scaffold composed ofany material and/or shape that (a) allows cells to attach to it (or canbe modified to allow cells to attach to it); and (b) allows cells togrow in more than one layer. This support is inoculated with stromalcells to form the living three-dimensional stromal tissue. The structureof the framework can include a mesh, a sponge or can be formed from ahydrogel.

Three-Dimensional Stromal Tissue or Living Stromal Matrix: athree-dimensional framework which has been inoculated with stromal cellsthat are grown on the support. The extracellular matrix proteinselaborated by the stromal cells are deposited onto the framework, thusforming a living stromal tissue. The living stromal tissue can supportthe growth of tissue-specific cells later inoculated to form thethree-dimensional cell culture.

Tissue-Specific Three-Dimensional Cell Culture or Tissue-SpecificThree-Dimensional Construct: a three-dimensional living stromal tissuewhich has been inoculated with tissue-specific cells and cultured. Ingeneral, the tissue specific cells used to inoculate thethree-dimensional stromal matrix should include the “stem” cells (or“reserve” cells) for that tissue; i.e., those cells which generate newcells that will mature into the specialized cells that form theparenchyma of the tissue.

The following abbreviations shall have the meanings indicated:

BCS=bovine calf serum

BFU-E=burst-forming unit-erythroid

TGF-β=transforming growth factor-β

CFU-C=colony forming unit-culture

CFU-GEMM=colony forming unit-granuloid, erythroid, monocyte,megakaryocyte

CSF=colony-stimulating factor

DMEM=Dulbecco's Modified Eagle's Medium

EDTA=ethylenediamine tetraacetic acid

FBS=fetal bovine serum

FGF=fibroblast growth factor

GAG=glycosaminoglycan

GM-CSF=granulocyte/macrophage colony-stimulating factor

HBSS=Hank's balanced salt solution

HS=horse serum

IGF=insulin-like growth factor

LTBMC=long term bone marrow culture

MEM=minimal essential medium

PBL=peripheral blood leukocytes

PBS=phosphate buffered saline

PDGF=platelet-derived growth factor

RPMI 1640=Roswell Park Memorial Institute medium number 1640 (GIBCO,Inc., Grand Island, N.Y.)

SEM=scanning electron microscopy

VEGF=vascular endothelial growth factor

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph representing the kinetics of the deposition ofglycosaminoglycans and collagen laid down over time by thethree-dimensional tissue products Transcyte™ and Dermagraft®. Thedeposition volumes of the glycosaminoglycans are dependent on the periodof growth while the deposition of collagen is not dependent on theperiod of growth.

FIG. 2 is a graph representing the effect of extracellular matrix(removed from Transcyte™) and added at dilutions of 1:2, 1:5, 1:10, and1:100 to monolayer cultures of human fibroblasts and keratinocytes. Themost significant effect illustrated is at a 1:10 dilution of the matrix.

FIG. 3 is a graph representing relative proliferation of humanfibroblasts and keratinocytes exposed to conditioned medium (cellculture medium which has previously supported the growth of cells inTranscyte™). An increase in cell response was revealed in as little asthree days.

FIG. 4 is a graph representing the effect of 1× conditioned medium (cellculture medium which has previously supported the growth of cells inTranscyte™) on collagen deposition of cells grown in three dimensions incomparison to base medium DMEM (containing 10% BLS supplemented mediumwith 2 mM L-glutamine and 1× antibiotic/antimycotic) supplemented with a1× final concentration of serum-free medium and medium. A statisticallysignificant (p=0.05) increase of almost 50% was noted in collagendeposition of cultures treated with the conditioned medium for 10 daysas compared with either control.

FIG. 5 is a graph representing the anti-oxidant activity in conditionedcell medium (cell culture medium which has previously supported thegrowth of cells in Transcyte™) on human epidermal keratinocytes inculture. A statistically significant (p<0.0003) reduction inintracellular oxidation of approximately 50% was noted in humankeratinocytes exposed to conditioned medium which had previouslysupported Transcyte™ for three days.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel compositions comprising anyconditioned defined or undefined medium, cultured using any eukaryoticcell type or three-dimensional tissue construct and methods for usingthe compositions. The cells are cultured in monolayer, beads (i.e.,two-dimensions) or, preferably, in three-dimensions. The cells arepreferably human to reduce the risk of an immune response and includestromal cells, parenchymal cells, mesenchymal stem cells, liver reservecells, neural stem cells, pancreatic stem cells and/or embryonic stemcells. Medium conditioned by cell and tissue cultures will contain avariety of naturally secreted proteins, such as biologically activegrowth factors and those cultured in three-dimensions will have theseproteins in ratios approaching physiological levels. The invention alsorelates to novel compositions comprising products derived from theconditioned cell media and uses for these compositions.

The “pre-conditioned” cell culture medium may be any cell culture mediumwhich adequately addresses the nutritional needs of the cells beingcultured. Examples of cell media include, but are not limited toDulbecco's Modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640,Iscove's, McCoy's and other media formulations readily apparent to thoseskilled in the art, including those found in Methods For Preparation ofMedia, Supplements and Substrate For Serum-Free Animal Cell Culture AlanR. Liss, New York (1984) and Cell & Tissue Culture: LaboratoryProcedures, John Wiley & Sons Ltd., Chichester, England 1996, both ofwhich are incorporated by reference herein in their entirety. The mediummay be supplemented, with any components necessary to support thedesired cell or tissue culture. Additionally serum, such as bovineserum, which is a complex solution of albumins, globulins, growthpromoters and growth inhibitors may be added if desired. The serumshould be pathogen free and carefully screened for mycoplasma bacterial,fungal, and viral contamination. Also, the serum should generally beobtained from the United States and not obtained from countries whereindigenous livestock carry transmittable agents. Hormone addition intothe medium may or may not be desired.

Other ingredients, such as vitamins, growth and attachment factors,proteins etc., can be selected by those of skill in the art inaccordance with his or her particular need. The present invention mayuse any cell type appropriate to achieve the desired conditioned medium.Genetically engineered cells may be used to culture the media. Suchcells can be modified, for example, to express a desired protein orproteins so that the concentration of the expressed protein or proteinsin the medium is optimized for the particular desired application. Inaccordance with the present invention, the cells and tissue culturesused to condition the medium may be engineered to express a target geneproduct which may impart a wide variety of functions, including but notlimited to, improved properties in expressing proteins resemblingphysiological reactions, increased expression of a particular proteinuseful for a specific application, such as wound healing or inhibitingcertain proteins such as proteases, lactic acid, etc.

The cells may be engineered to express a target gene product which isbiologically active which provides a chosen biological function, whichacts as a reporter of a chosen physiological condition, which augmentsdeficient or defective expression of a gene product, or which providesan anti-viral, anti-bacterial, anti-microbial, or anti-cancer activity.In accordance with the present invention, the target gene product may bea peptide or protein, such as an enzyme, hormone, cytokine, antigen, orantibody, a regulatory protein, such as a transcription factor or DNAbinding protein, a structural protein, such as a cell surface protein,or the target gene product may be a nucleic acid such as a ribosome orantisense molecule. The target gene products include, but are notlimited to, gene products which enhance cell growth. For example, thegenetic modification may upregulate an endogenous protein, introduce anew protein or regulate ion concentration by expressing a heterologousion channel or altering endogenous ion channel function. Examplesinclude, but are not limited to engineered tissues that express geneproducts which are delivered systemically (e.g., secreted gene productssuch as proteins including growth factors, hormones, Factor VIII, FactorIX, neurotransmitters, and enkaphalins).

In the present invention, it is preferred that the cells are grown on athree-dimensional stromal support and grow in multiple layers, forming acellular matrix. This matrix system approaches physiologic conditionsfound in vivo to a greater degree than previously described monolayertissue culture systems. Three-dimensional cultures, such as Dermagraft®(Advanced Tissue Sciences, Inc., La Jolla, Calif.) “Dermagraft®”, andTransCyte™ (Smith & Nephew, PLC, United Kingdom) “Transcyte™”, producenumerous growth factors and other proteins that are secreted into themedium at physiological ratios and concentrations. Dermagraft® iscomposed of allogeneic neonatal fibroblasts cultured on biodegradablepolyglactin. TransCyte™ is a temporary living skin replacementcomprising a three-dimensional stromal tissue bonded to a transitionalcovering as described in U.S. Pat. No. 5,460,939. Additionally, thethree-dimensional tissue cultures which condition the cell media maycontain mesenchymal stem cells, liver reserve cells, neural stem cells,pancreatic stem cells, and/or embryonic stem cells and/or parenchymalcells and/or parenchymal stem cells found in many tissue types,including but not limited to bone marrow, skin, liver, pancreas, kidney,adrenal and neurologic tissue, as well as tissues of thegastrointestinal and genitourinary tracts, and the circulatory system.See U.S. Pat. Nos. 4,721,096; 4,963,489; 5,032,508; 5,266,480;5,160,490; and 5,559,022, each of which is incorporated by referenceherein in their entirety.

Cell Media

Cell culture media formulations are well known in the literature andmany are commercially available.

Preconditioned media ingredients include, but are not limited to thosedescribed below. Additionally, the concentration of the ingredients iswell known to one of ordinary skill in the art. See, for example,Methods For Preparation Of Media, Supplements and Substrate forSerum-free Animal Cell Cultures, supra. The ingredients includeamino-acids (both D and/or L-amino acids) such as glutamine, alanine,arginine, asparagine, cysteine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine and their derivatives; acidsoluble subgroups such as thiamine, ascorbic acid, ferric compounds,ferrous compounds, purines, glutathione and monobasic sodium phosphates.

Additional ingredients include sugars, deoxyribose, ribose, nucleosides,water soluble vitamins, riboflavin, salts, trace metals, lipids, acetatesalts, phosphate salts, HEPES, phenol red, pyruvate salts and buffers.

Other ingredients often used in media formulations include fat solublevitamins (including A, D, E and K) steroids and their derivatives,cholesterol, fatty acids and lipids Tween 80, 2-mercaptoethanolpyramidines as well as a variety of supplements including serum (fetal,horse, calf, etc.), proteins (insulin, transferrin, growth factors,hormones, etc.) antibiotics (gentamicin, penicillin, streptomycin,amphotericin B, etc.) whole egg ultra filtrate, and attachment factors(fibronectins, vitronectins, collagens, laminins, tenascins, etc.).

Of course, the media may or may not need to be supplemented with growthfactors and other proteins such as attachment factors since many of thecell constructs, particularly the three-dimensional cell and tissueculture constructs described in this application themselves elaboratesuch growth and attachment factors and other products into the media asdiscussed in greater detail infra, Section 5.8.

The Cell Cultures The Cells

The medium may be conditioned by stromal cells, parenchymal cells,mesenchymal stem cells (lineage committed or uncommitted progenitorcells), liver reserve cells, neural stem cells, pancreatic stem cells,and/or embryonic stem cells. The cells may include, but are not limitedto, bone marrow, skin, liver, pancreas, kidney, neurological tissue,adrenal gland, mucosal epithelium, and smooth muscle, to name but a few.The fibroblasts and fibroblast-like cells and other cells and/orelements that comprise the stroma may be fetal or adult in origin, andmay be derived from convenient sources such as skin, liver, pancreas,mucosa, arteries, veins, umbilical cord, and placental tissues, etc.Such tissues and/or organs can be obtained by appropriate biopsy or uponautopsy. In fact, cadaver organs may be used to provide a generoussupply of stromal cells and elements.

Embryonic stem cells and/or other elements that comprise the stroma maybe isolated using methods known in the art. For instance, recently humanembryonic stem cell populations and methods for isolating and usingthese cells have been reported in Keller et al., Nature Med., 5:151-152(1999), Smith Curr. Biol. 8:R802-804 (1998); isolated from primordialgerm cells, Shamblatt et al., PNAS 95:13726-1373 (1998), isolated fromblastocytes Thomason et al., Science 282:1145-1147 (1988). The isolationand culture of mesenchymal stem cells are known in the art. See Mackayet al., Tissue Eng. 4:415-428 (1988); William et al., Am Surg. 65:22-26(1999). Inoculation of these cells is described infra, in Section 5.3.Likewise, neural stem cells may be isolated in the manner described inFlax et al., Nature Biotechnol., 16:1033-1039 (1998); and Frisen et al.,Cell. Mol. Life Sci., 54:935-945 (1998).

The cells may be cultured in any manner known in the art including inmonolayer, beads or in three-dimensions and by any means (i.e., culturedish, roller bottle, a continuous flow system, etc.). Methods of celland tissue culturing are well known in the art, and are described, forexample, in Cell & Tissue Culture: Laboratory Procedures, supra,Freshney (1987), Culture of Animal Cells: A Manual of Basic Techniques,infra.

In general, the cell lines utilized are carefully screened for human andanimal pathogens. Depending upon the application, such screening may beof critical importance where only pathogen free cells are acceptable(e.g., for wound healing, food additives, etc.) Methods of screening forpathogens are well known in the art. The cell type, whether cultured intwo-dimensions or three-dimensions, will affect the properties of theconditioned medium. A three-dimensional construct is preferred.

Three-Dimensional Cell Cultures

The stromal cells used in the three-dimensional cultures comprisefibroblasts, mesenchymal stem cells, liver reserve cells, neural stemcells, pancreatic stem cells, and/or embryonic stem cells with orwithout additional cells and/or elements described more fully herein.

Fibroblasts will support the growth of many different cells and tissuesin the three-dimensional culture system, and, therefore, can beinoculated onto the matrix to form a “generic” stromal support matrixfor culturing any of a variety of cells and tissues. However, in certaininstances, it may be preferable to use a “specific” rather than“generic” stromal support matrix, in which case stromal cells andelements can be obtained from a particular tissue, organ, or individual.Moreover, fibroblasts and other stromal cells and/or elements may bederived from the same type of tissue to be cultured in thethree-dimensional system. This might be advantageous when culturingtissues in which specialized stromal cells may play particularstructural/functional roles; e.g., smooth muscle cells of arteries,glial cells of neurological tissue, Kupffer cells of liver, etc.

Once inoculated onto the three-dimensional support, the stromal cellswill proliferate on the framework and deposit the connective tissueproteins naturally secreted by the stromal cells such as growth factors,regulatory factors and extracellular matrix proteins. The stromal cellsand their naturally secreted connective tissue proteins substantiallyenvelop the framework thus forming the living stromal tissue which willsupport the growth of tissue-specific cells inoculated into thethree-dimensional culture system of the invention. In fact, wheninoculated with the tissue-specific cells, the three-dimensional stromaltissue will sustain active proliferation of the culture for long periodsof time. Importantly, because openings in the mesh permit the exit ofstromal cells in culture, confluent stromal cultures do not exhibitcontact inhibition, and the stromal cells continue to grow, divide, andremain functionally active.

Growth and regulatory factors are elaborated by the stromal tissue intothe media. Growth factors (for example, but not limited to, αFGF, βFGF,insulin growth factor or TGF-betas), or natural or modified bloodproducts or other bioactive biological molecules (for example, but notlimited to, hyaluronic acid or hormones), enhance the colonization ofthe three-dimensional framework or scaffolding and condition the culturemedia.

The extent to which the stromal cells are grown prior to use of thecultures in vivo may vary depending on the type of tissue to be grown inthree-dimensional tissue culture. The living stromal tissues whichcondition the medium may be used as corrective structures by implantingthem in vivo. Alternatively, the living stromal tissues may beinoculated with another cell type and implanted in vivo. The stromalcells may be genetically engineered to adjust the level of proteinproducts secreted into the culture medium to improve the concentrationof recovered product obtained from the conditioned medium. For example,anti-inflammatory factors, e.g., anti-GM-CSF, anti-TNF, anti-IL-1,anti-IL-2, etc. Alternatively, the stromal cells may be geneticallyengineered to “knock out” expression of native gene products thatpromote inflammation, e.g., GM-CSF, TNF, IL-1, IL-2, or “knock out”expression of MHC in order to lower the risk of rejection.

Growth of the stromal cells in three-dimensions will sustain activeproliferation of both the stromal and tissue-specific cells in culturefor much longer time periods than will monolayer systems. Moreover, thethree-dimensional system supports the maturation, differentiation, andsegregation of cells in culture in vitro to form components of adulttissues analogous to counterparts found in vivo and secure proteins intothe conditional medium more closely resembling physiological ratios.

Although the Applicants are under no duty or obligation to explain themechanism by which the three-dimensional cell and tissue and the works,a number of factors inherent in the three-dimensional culture system maycontribute to its success:

(a) The three-dimensional framework provides a greater surface area forprotein attachment, and consequently, for the adherence of stromalcells; and

(b) Because of the three-dimensionality of the framework, stromal cellscontinue to grow actively, in contrast to cells in monolayer cultures,which grow to confluence, exhibit contact inhibition, and cease to growand divide. The elaboration of growth and regulatory factors byreplicating stromal cells may be partially responsible for stimulatingproliferation and regulating differentiation of cells in culture;

(c) The three-dimensional framework allows for a spatial distribution ofcellular elements which is more analogous to that found in thecounterpart tissue in vivo;

(d) The increase in potential volume for cell growth in thethree-dimensional system may allow the establishment of localizedmicroenvironments conducive to cellular maturation;

(e) The three-dimensional framework maximizes cell-cell interactions byallowing greater potential for movement of migratory cells, such asmacrophages, monocytes and possibly lymphocytes in the adherent layer;

(f) It has been recognized that maintenance of a differentiated cellularphenotype requires not only growth/differentiation factors but also theappropriate cellular interactions. The present invention effectivelyrecreates the tissue microenvironment resulting in a superiorconditioned medium.

Establishment of Three-Dimensional Stromal Tissue

The three-dimensional support or framework may be of any material and/orshape that: (a) allows cells to attach to it (or can be modified toallow cells to attach to it); and (b) allows cells to grow in more thanone layer. A number of different materials may be used to form theframework, including but not limited to: non-biodegradable materials,e.g., nylon (polyamides), dacron (polyesters), polystyrene,polypropylene, polyacrylates, polyvinyl compounds (e.g.,polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE;teflon), thermanox (TPX), nitrocellulose, cotton; and biodegradablematerials, e.g., polyglycolic acid (PGA), collagen, collagen sponges,cat gut sutures, cellulose, gelatin, dextran, polyalkanoates, etc. Anyof these materials may be woven braided, knitted, etc., into a mesh, forexample, to form the three-dimensional framework. The framework, in tumcan be fashioned into any shape desired as the corrective structure,e.g., tubes, ropes, filaments, etc. Certain materials, such as nylon,polystyrene, etc., are poor substrates for cellular attachment. Whenthese materials are used as the three-dimensional framework, it isadvisable to pre-treat the framework prior to inoculation of stromalcells in order to enhance the attachment of stromal cells to thesupport. For example, prior to inoculation with stromal cells, nylonframeworks could be treated with 0.1M acetic acid, and incubated inpolylysine, FBS, and/or collagen to coat the nylon. Polystyrene could besimilarly treated using sulfuric acid.

When the cultures conditioning the medium are to be implanted in vivo,it may be preferable to use biodegradable matrices such as polyglycolicacid, collagen, collagen sponges, woven collagen, catgut suturematerial, gelatin, polylactic acid, or polyglycolic acid and copolymersthereof, for example. Where the cultures are to be maintained for longperiods of time or cryopreserved, non-degradable materials such asnylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, cotton,etc., may be preferred. A convenient nylon mesh which could be used inaccordance with the invention is Nitex, a nylon filtration mesh havingan average pore size of 210 μm and an average nylon fiber diameter of 90μm (#3-210/36, Tetko, Inc., N.Y.).

Stromal cells comprising fibroblasts, mesenchymal stem cells liverreserve cells, neural stem cells, pancreatic stem cells and/or embryonicstem cells with or without other cells and elements described below, areinoculated onto the framework. Also, cells found in loose connectivetissue may be inoculated onto the three-dimensional support along withfibroblasts. Such cells include but are not limited to smooth musclecells, endothelial cells, pericytes, macrophages, monocytes, plasmacells, mast cells, adipocytes, etc. As previously explained, fetalfibroblasts can be used to form a “generic” three-dimensional stromalmatrix that will support the growth of a variety of different cellsand/or tissues. However, a “specific” stromal tissue may be prepared byinoculating the three-dimensional framework with fibroblasts derivedfrom the same type of tissue to be cultured and/or from a particularindividual who is later to receive the cells and/or tissues grown inculture in accordance with the three-dimensional system of theinvention.

Thus, in one embodiment of the invention, stromal cells which arespecialized for the particular tissue may be cultured. For example,stromal cells of hematopoietic tissue, including but not limited tofibroblasts, endothelial cells, macrophages/monocytes, adipocytes andreticular cells, could be used to form the three-dimensionalsubconfluent stroma for the long term culture of bone marrow in vitro.Hematopoietic stromal cells may be readily obtained from the “buffycoat” formed in bone marrow suspensions by centrifugation at low forces,e.g., 3000×g. In the stromal layer that makes up the inner wall ofarteries, a high proportion of undifferentiated smooth muscle cells canbe added to provide the protein elastic. Stromal cells of liver mayinclude fibroblasts, Kupffer cells, and vascular and bile ductendothelial cells. Similarly, glial cells could be used as the stroma tosupport the proliferation of neurological cells and tissues; glial cellsfor this purpose can be obtained by trypsinization or collagenasedigestion of embryonic or adult brain (Ponten and Westermark, 1980, inFederof, S. Hertz, L., eds, “Advances in Cellular Neurobiology,” Vol. 1,New York, Academic Press, pp. 209-227). The growth of cells in thethree-dimensional stromal cell culture may be further enhanced by addingto the framework, or coating the support with proteins (e.g., collagens,elastic fibers, reticular fibers) glycoproteins, glycosaminoglycans(e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate,dermatan sulfate, keratin sulfate, etc.), a cellular matrix, and/orother materials.

Further, mesenchymal stem cells (lineage committed or uncommittedprogenitor cells) are advantageous “stromal” cells for inoculation ontothe framework. The cells may differentiate into osteocytes, fibroblastsof the tendons and ligaments, marrow stromal cells, adipocytes and othercells of connective tissue, chondrocytes, depending of course, onendogens or supplemented growth and regulatory factors and other factorsincluding prostaglandin, interleukins and naturally—occurring chaloneswhich regulate proliferation and/or differentiation.

Fibroblasts may be readily isolated by disaggregating an appropriateorgan or tissue which is to serve as the source of the fibroblasts. Thismay be readily accomplished using techniques known to those skilled inthe art. For example, the tissue or organ can be disaggregatedmechanically and/or treated with digestive enzymes and/or chelatingagents that weaken the connections between neighboring cells making itpossible to disperse the tissue into a suspension of individual cellswithout appreciable cell breakage. Enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withany of a number of digestive enzymes either alone or in combination.These include but are not limited to trypsin, chymotrypsin, collagenase,elastase, and/or hyaluronidase, DNase, pronase, dispase etc. Mechanicaldisruption can also be accomplished by a number of methods including,but not limited to, the use of grinders, blenders, sieves, homogenizers,pressure cells, or insonators to name but a few. For a review of tissuedisaggregation techniques, see Freshney, Culture of Animal Cells: AManual of Basic Technique, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch.9, pp. 107-126.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thefibroblasts and/or other stromal cells and/or elements can be obtained.This also may be accomplished using standard techniques for cellseparation including, but not limited to, cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counterstreaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting. For a review of clonal selection and cell separationtechniques, see Freshney, Culture of Animal Cells: A Manual of BasicTechniques, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137-168.

The isolation of fibroblasts may, for example, be carried out asfollows: fresh tissue samples are thoroughly washed and minced in Hanksbalanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated fibroblasts can then be grown to confluency, liftedfrom the confluent culture and inoculated onto the three-dimensionalmatrix (see, Naughton et al., 1987, J Med. 18 (3 and 4) 219-250).Inoculation of the three-dimensional framework with a high concentrationof stromal cells, e.g., approximately 10⁶ to 5×10⁷ cells/ml, will resultin the establishment of the three-dimensional stromal tissue in shorterperiods of time.

After inoculation of the stromal cells, the three-dimensional frameworkshould be incubated in an appropriate nutrient medium. As previouslymentioned, many commercially available media such as RPMI 1640,Fisher's, Iscove's, McCoy's, and the like may be suitable for use. It isimportant that the three-dimensional stromal cell cultures be suspendedor floated in the medium during the incubation period in order tomaximize proliferative activity. The culture is “fed” periodically andthe conditioned media of the invention is recovered and processed asdescribed below in Sections 5.6 and 5.7. Thus, depending upon the tissueto be cultured and the collagen types desired, the appropriate stromalcell(s) may be selected to inoculate the three-dimensional matrix.

During incubation of the three-dimensional stromal cell cultures,proliferating cells may be released from the matrix. These releasedcells may stick to the walls of the culture vessel where they maycontinue to proliferate and form a confluent monolayer. This should beprevented or minimized, for example, by removal of the released cellsduring feeding, or by transferring the three-dimensional stromal cultureto a new culture vessel. The presence of a confluent monolayer in thevessel will “shut down” the growth of cells in the three-dimensionalmatrix and/or culture. Removal of the confluent monolayer or transfer ofthe culture to fresh media in a new vessel will restore proliferativeactivity of the three-dimensional culture system. It should be notedthat the conditioned media of the invention is processed, if necessary,so that it does not contain any whole cells (unless of course, wholecells are used for a specific application). Such removal or transfersshould be done in any culture vessel which has a stromal monolayerexceeding 25% confluency. Alternatively, the culture system could beagitated to prevent the released cells from sticking, or instead ofperiodically feeding the cultures, the culture system could be set up sothat fresh media continuously flows through the system. The flow ratecould be adjusted to both maximize proliferation within thethree-dimensional culture, and to wash out and remove cells releasedfrom the culture, so that they will not stick to the walls of the vesseland grow to confluence.

Other cells, such as parenchymal cells, may be inoculated and grown onthe three-dimensional living stromal tissue.

Inoculation of Tissue-Specific Cells onto Three-Dimensional StromalMatrix and Maintenance of Cultures

Once the three-dimensional stromal cell culture has reached theappropriate degree of growth, additional cells such as tissue-specificcells (parenchymal cells) or surface layer cells which are desired to becultured may also be inoculated onto the living stromal tissue. Thecells are grown on the living stromal tissue in vitro to form a culturedcounterpart of the native tissue and condition the media by elaboratingextracellular products into the media at ratios resembling physiologicallevels. A high concentration of cells in the inoculum willadvantageously result in increased proliferation in culture much soonerthan will low concentrations. The cells chosen for inoculation willdepend upon the tissue to be cultured, which may include, but is notlimited to, bone marrow, skin, liver, pancreas, kidney, neurologicaltissue, adrenal gland, mucosal epithelium, and smooth muscle, to namebut a few. Such cells with elaborate characteristic extracellularproteins such as certain growth factors into the media resulting inmedia optimized for certain tissue specific applications.

For example, and not by way of limitation, a variety of epithelial cellscan be cultured on the three-dimensional living stromal tissue. Examplesof such epithelial cells include, but are not limited to, keratinocytes,oral mucosa and gastrointestinal (G.I.) tract cells. Such epithelialcells may be isolated by enzymatic treatment of the tissue according tomethods known in the art, followed by expansion of these cells inculture and application of epithelial cells to the three-dimensionalstromal support cell matrix. The presence of the stromal supportprovides growth factors and other proteins which promote normal divisionand differentiation of epithelial cells.

In general, this inoculum should include the “stem” cell (also calledthe “reserve” cell) for that tissue; i.e., those cells which generatenew cells that will mature into the specialized cells that form thevarious components of the tissue.

The parenchymal or other surface layer cells used in the inoculum may beobtained from cell suspensions prepared by disaggregating the desiredtissue using standard techniques described for obtaining stromal cellsin Section 5.3 above. The entire cellular suspension itself could beused to inoculate the three-dimensional living stromal tissue. As aresult, the regenerative cells contained within the homogenate willproliferate, mature, and differentiate properly on the matrix, whereasnon-regenerative cells will not. Alternatively, particular cell typesmay be isolated from appropriate fractions of the cellular suspensionusing standard techniques described for fractionating stromal cells inSection 5.1 above. Where the “stem” cells or “reserve” cells can bereadily isolated, these may be used to preferentially inoculate thethree-dimensional stromal support. For example, when culturing bonemarrow, the three-dimensional stroma may be inoculated with bone marrowcells, either fresh or derived from a cryopreserved sample. Whenculturing skin, the three-dimensional stroma may be inoculated withmelanocytes and keratinocytes. When culturing liver, thethree-dimensional stroma may be inoculated with hepatocytes. Whenculturing pancreas, the three-dimensional stroma may be inoculated withpancreatic endocrine cells. For a review of methods which may beutilized to obtain parenchymal cells from various tissues, see,Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed.,A.R. Liss, Inc., New York, 1987, Ch. 20, pp. 257-288.

In fact, different proportions of the various types of collagendeposited on the stromal matrix prior to inoculation can affect thegrowth of the later-inoculated tissue-specific cells. For example, foroptimal growth of hematopoietic cells, the matrix should preferablycontain collagen types III, IV and I in an approximate ratio of 6:3:1 inthe initial matrix. For three dimensional skin culture systems, collagentypes I and III are preferably deposited in the initial matrix. Theproportions of collagen types deposited can be manipulated or enhancedby selecting fibroblasts which elaborate the appropriate collagen type.This can be accomplished using monoclonal antibodies of an appropriateisotype or subclass that is capable of activating complement, and whichdefine particular collagen types. These antibodies and complement can beused to negatively select the fibroblasts which express the desiredcollagen type. Alternatively, the stromal cells used to inoculate thematrix can be a mixture of cells which synthesize the appropriatecollagen type desired. The distribution and origins of various types ofcollagen is shown in Table I.

TABLE 1 DISTRIBUTIONS AND ORIGINS OF VARIOUS TYPES OF COLLAGEN CollagenType Principal Tissue Distribution Cells of Origin I Loose and denseordinary Fibroblasts and reticular cells; connective tissue; collagensmooth muscle cells fibers Fibrocartilage Bone Osteoblasts DentinOdontoblasts II Hyaline and elastic cartilage Chondrocytes Vitreous bodyof eye Retinal cells III Loose connective tissue; Fibroblasts andreticular cells reticular fibers Papillary layer of dermis Blood vesselsSmooth muscle cells; endothelial cells IV Basement membranes Epithelialand endothelial cells Lens capsule of eye Lens fibers V Fetal membranes;placenta Fibroblast Basement membranes Bone Smooth muscle Smooth musclecells Fibroblasts VI Connective Tissue VII Epithelial basementFibroblasts, keratinocytes membranes, anchoring fibrils Cornea Cornealfibroblasts VIII Cartilage IX Hypertrophic cartilage X CartilageFibroblasts XI Papillary dermis Fibroblasts XII Reticular dermisFibroblasts XIV P 170 bullous pemphigoid Keratinocytes antigen undulinXVII

During incubation, the three-dimensional cell culture system should besuspended or floated in the nutrient medium. Cultures should be fed withfresh media periodically. Again, care should be taken to prevent cellsreleased from the culture from sticking to the walls of the vessel wherethey could proliferate and form a confluent monolayer. The release ofcells from the three-dimensional culture appears to occur more readilywhen culturing diffuse tissues as opposed to structured tissues. Forexample, the three-dimensional skin culture of the invention ishistologically and morphologically normal; the distinct dermal andepidermal layers do not release cells into the surrounding media. Bycontrast, the three-dimensional bone marrow cultures of the inventionrelease mature non-adherent cells into the medium much the way suchcells are released in marrow in vivo. As previously explained, shouldthe released cells stick to the culture vessel and form a confluentmonolayer, the proliferation of the three-dimensional culture will be“shut down”. This can be avoided by removal of released cells duringfeeding, transfer of the three-dimensional culture to a new vessel, byagitation of the culture to prevent sticking of released cells to thevessel wall, or by the continuous flow of fresh media at a ratesufficient to replenish nutrients in the culture and remove releasedcells. As previously mentioned, the conditioned media is processed, ifnecessary, so that it is free of whole cells and cellular debris.

The growth and activity of cells in culture can be affected by a varietyof growth factors such as insulin, growth hormone, somatomedins, colonystimulating factors, erythropoietin, epidermal growth factor, hepaticerythropoietic factor (hepatopoietin), and liver-cell growth factor.Other factors which regulate proliferation and/or differentiationinclude prostaglandins, interleukins, and naturally-occurring chalones.

Genetically Engineered Constructs

In another embodiment, the three-dimensional constructs conditioning themedia can act as vehicles for introducing gene products into the mediathat promote the repair and/or regeneration of tissue defects, forexample. The cells can be genetically engineered to express, forexample, inflammatory mediators, such as IL-6, IL-8 and G-CSF. The cellscould also or alternatively be genetically engineering to expressanti-inflammatory factors, e.g., anti-GM-CSF, anti-TNF, anti-IL-1,anti-IL-2, etc.

In another embodiment, the cells can be genetically engineered toexpress a gene into the media which would exert a therapeutic effect,e.g., in the production of TGF-to stimulate cartilage production, orother factors such as BMP-13 to promote chondrogenesis or stimulatoryfactors that promote migration of stromal cells and/or matrixdeposition. Since the constructs comprise eukaryotic cells, the geneproduct will be properly expressed and processed to form an activeproduct. Preferably, the expression control elements used should allowfor the regulated expression of the gene so that the product can beover-synthesized in culture. The transcriptional promoter chosen,generally, and promoter elements specifically, depend, in part, upon thetype of tissue and cells cultured. Cells and tissues which are capableof secreting proteins are preferable (e.g., those having abundant roughendoplasmic reticulum and Golgi complex organelles). The over-producedgene product will then be secreted by the engineered cell into theconditioned media.

The cells used to condition the media may be genetically engineered toregulate one or more genes; or the regulation of gene expression may betransient or long-term; or the gene

activity may be non-inducible or inducible. The cells that condition themedia can also be genetically engineered to “knock out” expression offactors that promote inflammation. Negative modulatory techniques forthe reduction of target gene expression levels or target gene productactivity levels are discussed below. “Negative modulation”, as usedherein, refers to a reduction in the level and/or activity of targetgene product relative to the level and/or activity of the target geneproduct in the absence of the modulatory treatment. The expression of agene native to the cell can be reduced or knocked out using a number oftechniques, for example, expression may be inhibited by inactivating thegene completely (commonly termed “knockout”) using standard homologousrecombination techniques. Usually, an exon encoding an important regionof the protein (or an exon 5′ to that region) is interrupted by apositive selectable marker (for example neo), preventing the productionof normal mRNA from the target gene and resulting in inactivation of thegene. A gene may also be inactivated by creating a deletion or aninactivating insertion in part of a gene, or by deleting the entiregene. By using a construct with two regions of homology to the targetgene that are far apart in the genome, the sequences intervening the tworegions can be deleted. Mombaerts et al., 1991, Proc. Nat. Acad. Sci.USA. 88:3084-3087. Alternatively, a gene may also be inactivated bydeletion of upstream or downstream expression elements.

Antisense and ribozyme molecules which inhibit expression of the targetgene can also be used in accordance with the invention to reduce thelevel of target gene activity. For example, antisense RNA moleculeswhich inhibit the expression of major histocompatibility gene complexes(HLA) have been shown to be most versatile with respect to immuneresponses. Furthermore, appropriate ribozyme molecules can be designedas described, e.g., by Haseloff et al., 1988, Nature 334:585-591; Zauget al., 1984, Science 224:574-578; and Zaug and Cech, 1986, Science231:470-475. Still further, triple helix molecules can be utilized inreducing the level of target gene activity. These techniques aredescribed in detail by L. G. Davis et al., eds, Basic Methods inMolecular Biology, 2nd ed., Appleton & Lange, Norwalk, Conn. 1994.

Methods that may be useful to genetically engineer the cells of theinvention are well-known in the art and are further detailed in co-ownedU.S. Pat. Nos. 4,963,489 and 5,785,964, the disclosures of which areincorporated herein by reference. For example, a recombinant DNAconstruct or vector containing an exogenous nucleic acid, e.g., encodinga gene product of interest, may be constructed and used to transform ortransfect the stromal cells of the invention. Such transformed ortransfected cells that carry the exogenous nucleic acid, and that arecapable of expressing said nucleic acid, are selected and clonallyexpanded in the three-dimensional constructs of this invention.

Methods for preparing DNA constructs containing the gene of interest,for transforming or transfecting cells, and for selecting cells carryingand expressing the gene of interest are well-known in the art. See, forexample, the techniques described in Maniatis et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Ausubel et al., 1989, Current Protocols inMolecular Biology, Greene Publishing Associates & Wiley Interscience,N.Y.; and Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The cells can be engineered using any of a variety of vectors including,but not limited to, integrating viral vectors, e.g., retrovirus vectoror adena-associated viral vectors; or non-integrating replicatingvectors, e.g., papilloma virus vectors, SV40 vectors, adenoviralvectors; or replication-defective viral vectors. Where transientexpression is desired, non-integrating vectors and replication defectivevectors may be preferred, since either inducible or constitutivepromoters can be used in these systems to control expression of the geneof interest. Alternatively, integrating vectors can be used to obtaintransient expression, provided the gene of interest is controlled by aninducible promoter. Other methods of introducing DNA into cells includethe use of liposomes, lipofection, electroporation, a particle gun, orby direct DNA injection.

The cells are preferably transformed or transfected with a nucleic acid,e.g., DNA, controlled by, i.e., in operative association with, one ormore appropriate expression control elements such as promoter orenhancer sequences, transcription terminators, polyadenylation sites,among others, and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow in enriched mediaand then switched to selective media. The selectable marker in theforeign DNA confers resistance to the selection and allows cells tostably integrate the foreign DNA as, for example, on a plasmid, intotheir chromosomes and grow to form foci which, in tum, can be cloned andexpanded into cell lines. This method can be advantageously used toengineer cell lines which express the gene product into the media.

Any promoter may be used to drive the expression of the inserted gene.For example, viral promoters include but are not limited to the CMVpromoter/enhancer, SV40, papillomavirus, Epstein-Barr virus, elastingene promoter and -globin. Preferably, the control elements used tocontrol expression of the gene of interest should allow for theregulated expression of the gene so that the product is synthesized onlywhen needed in vivo. If transient expression is desired, constitutivepromoters are preferably used in a non-integrating and/orreplication-defective vector. Alternatively, inducible promoters couldbe used to drive the expression of the inserted gene when necessary.Inducible promoters can be built into integrating and/or replicatingvectors. For example, inducible promoters include, but are not limitedto, metallothionien and heat shock protein.

According to one embodiment, the inducible promoters used for expressingexogenous genes of interest are those that are the native promoters ofthose regulatory proteins as disclosed herein that are induced as aresult of cyropreservation and subsequent thawing. For example, thepromoter of TGF-β, VEGF, or various known heat shock proteins can beused as the expression control element, i.e., can be operatively linkedto an exogenous gene of interest in order to express a desired geneproduct in the tissue constructs conditioning the cell media.

A variety of methods may be used to obtain the constitutive or transientexpression of gene products engineered into the cells. For example, thetranskaryotic implantation technique described by Seldon et al., 1987,Science 236:714-718 can be used. “Transkaryotic”, as used herein,suggests that the nuclei of the implanted cells have been altered by theaddition of DNA sequences by stable or transient transfection.Preferably, the cells are engineered to express such gene productstransiently and/or under inducible control during the post-operativerecovery period, or as a chimeric fusion protein anchored to the stromalcells, for example, as a chimeric molecule composed of an intracellularand/or transmembrane domain of a receptor or receptor-like molecule,fused to the gene product as the extracellular domain.

Furthermore, it may be desirable to prepare a construct having anextracellular matrix containing a foreign gene product, growth factor,regulatory factor, etc., which is then found in the conditioned media.This embodiment is based on the discovery that, during the growth ofhuman stromal cells on a three-dimensional support framework, the cellssynthesize and deposit on the framework a human extracellular matrix asproduced in normal human tissue. The extracellular matrix is secretedlocally by cells and not only binds cells and tissue together but alsoinfluences the development and behavior of the cells it contacts. Theextracellular matrix contains various connective tissue proteins, e.g.,fiber-forming proteins interwoven in a hydrated gel composed of anetwork of glycosaminoglycans chains. The glycosaminoglycans are aheterogeneous group of long, negatively charged polysaccharide chains,which (except for hyaluronic acid) are covalently linked to protein toform proteoglycans molecules. According to this embodiment of theinvention, the stromal cells may be genetically engineered to express adesired gene product, or altered forms of a gene product, which will bepresent in the extracellular matrix and ultimately the cell medium.

Recovery of the Conditioned Media

The cells can be cultured by any means known in the art. Preferably, thecells are cultured in an environment which enables aseptic processingand handling. Conventional means of cell and tissue culture have beenlimited by the need for human supervision and control of the media. Thislimits the amount of cells and tissue that can be cultured at a singletime and consequently the volume of conditioned cell media that can beobtained at a single time. For this reason, it is preferred that themedia be conditioned in a manner allowing for large scale growth(yielding large scale conditioned media) using, for example, anapparatus for aseptic large scale culturing like that described inco-owned U.S. Pat. No. 5,763,267 (the '267 patent) which is incorporatedby reference herein in its entirety for all purposes. Using the asepticclosed system described in the '267 patent, preconditioned culture mediais transported from a fluid reservoir to an inlet manifold and evenlydistributed to the cultures in a continuous flow system and is useful inculturing three-dimensional cell and tissue cultures, such asDermagraft® for example. In particular, the apparatus described in the'267 patent includes a plurality of flexible or semi-flexible treatmentchambers comprising one or more individual culture pockets, a pluralityof rigid spacers, an inlet fluid manifold, an outlet fluid manifold, afluid reservoir, and a means for transporting fluid within the system.

During treatment, liquid medium is transported from the fluid reservoirto the inlet manifold, which in tum evenly distributes the media to eachof the connected treatment chambers and internal culture pockets. Anoutlet fluid manifold is also provided to ensure that each treatmentchamber is evenly filled and to ensure that any air bubbles formedduring treatment are removed from the treatment chambers. The treatmentchambers are flexible or semi-flexible so as to provide for easyend-user handling during rinsing and application of the culturedtransplants. Due to the flexibility of the treatment chambers, rigidspacers are also provided which ensure even fluid distribution withinthe chambers during treatment. When appropriate (i.e., once the mediumis conditioned so that extracellular proteins such as growth factorshave reached desirable levels in the medium) the “condition” medium ispumped out of the system and processed for use. Preferably, theconditioned cell medium is harvested from the apparatus at the laterstages of growth of the tissue when the level of certain growth factorsand connective tissue protein secretion is at its highest level (SeeFIG. 1). In a preferred embodiment, the medium conditioned by the threedimensional cell culture is collected after exposure of the medium tothe cells at days 10 through day 14 of culturing.

In another embodiment, the three-dimensional tissue is cultivated in anapparatus for aseptic growth of three-dimensional tissue cultures asdescribed in U.S. Pat. No. 5,843,766 (the '766 patent) incorporatedherein in its entirety for all purposes. The '766 patent discloses atissue culture chamber in which the chamber is a casing that providesfor growth of three-dimensional tissue that can be grown, preserved infrozen form, and shipped to the end user in the same aseptic container.The tissue culture chamber includes a casing comprising a substratewithin the casing designed to facilitate three-dimensional tissue growthon the surface of the substrate. The casing includes an inlet and anoutlet port which assist the inflow and outflow of medium. The casingalso includes at least one flow distributor. In one embodiment, the flowdistributor is a baffle, which is used to distribute the flow of themedium within the chamber to create a continuous, uniform piece ofthree-dimensional tissue. In a second embodiment, the flow distributoris a combination of deflector plates, distribution channels, and a flowchannel. In each embodiment, the casing further includes a seal so as toensure an aseptic environment inside the chamber during tissue growthand storage. Again the medium is preferably harvested from the apparatusat the later stages of growth of the tissue when the level of in growthfactors and connective tissue protein secretion is at its highest level(See FIG. 1). In a preferred embodiment, the medium conditioned by thethree dimensional cell culture is collected after exposure of the mediumto the cells at days 10 through day 14 of culturing.

Concentration of the Conditioned Medium

Following removal of the cell conditioned medium, it may be necessary tofurther process the resulting supernatant. Such processing may include,but are not limited to, concentration by a water flux filtration deviceor by defiltration using the methods described in Cell & Tissue Culture:Laboratory Procedures, supra, pp 29 D:0.1-29D:0.4.

Additionally, the medium may be concentrated 10 to 20 fold using apositive pressure concentration device having a filter with a 10,000 mlcut-off (Amicon, Beverly, Mass.).

Also, the conditioned medium may be further processed for productisolation and purification to remove unwanted proteases, for example.The methods used for product isolation and purification so that optimalbiological activity is maintained will be readily apparent to one ofordinary skill in the art. For example, it may be desirous to purify agrowth factor, regulatory factor, peptide hormone, antibody, etc. Suchmethods include, but are not limited to, gel chromatography (usingmatrices such as sephadex) ion exchange, metal chelate affinitychromatography with an insoluble matrix such as cross-linked agarose,HPLC purification and hydrophobic interaction chromatography of theconditioned media. Such techniques are described in greater detail inCell & Tissue Culture: Laboratory Procedures, supra. Of course,depending upon the desired application of the conditioned medium, and/orproducts derived thereof, appropriate measures must be taken to maintainsterility. Alternatively, sterilization may be necessary and can beaccomplished by methods known to one of ordinary skill in the art, suchas, for example, heat and/or filter sterilization taking care topreserve the desired biological activity.

Isolation of Collagen

As previously mentioned, the conditioned medium of the inventioncontains numerous products which may be isolated and purified therefrom.For example, human dermal fibroblasts synthesize and secrete collagenprecursors and a fraction of these precursors are incorporated into athree-dimensional extracellular matrix. This incorporation requires theremoval of terminal peptides (N- and C-peptides) which significantlylowers the solubility of the collagen molecules (the rest of thesecreted collagen remains in solution due to lack of proteolysis).Generally, soluble collagen may be obtained under neutral pH conditionsat high salt concentrations. See Kielty, C. M., I. Hopkinson, et al.(1993), Collagen: The Collagen Family: Structure, Assembly, andOrganization in the Extracellular Matrix, Connective Tissue and ItsHeritable Disorders: molecular, genetic and medical aspects. P. M. Royceand B. Steinmann. New York, Wiley-Liss, Inc.: 103-149). Applicantsprovide data showing the effect of conditioned medium (medium which haspreviously supported the growth of cells cultured in three dimensions)on the preparation and composition of three-dimensional tissues bymeasuring the amount of collagen secreted into the extracellular matrixof tissues cultured in the presence of serum-free medium, medium orthree dimensional conditioned medium (see Section 6.3). The conditionedmedium of the invention significantly increases collagen deposition oftissue in vitro as shown in FIG. 4

Further, Applicants' have discovered that, surprisingly, collagen is notlaid down in linear fashion, but is instead secreted at increasinglevels during the culturing process (see FIG. 1). Accordingly,Applicants' have applied this discovery when harvesting the collagen.

It should be understood that the following protocol is offered by way ofexample and may be modified using methods known to those of skill in therelevant art. To purify the collagen, add 240 mL of medium conditionedwith fibroblasts to 240 mL 5M NaCl (a 1:1 ratio of medium to salt) andprecipitate for 16 hours at 4° Celsius. Centrifuge the suspension forapproximately 20 minutes at 4000×g. Discard the supernatant. Wash thepellet with 10 mL of a solution of 50 mM Tris-HCl (pH 7.5) and 2.4MNaCl. Centrifuge for 20 minutes at 4000×g and discard the supernatant.Resuspend the pellet in 10 mL of 0.5M acetic acid. To remove thepropeptides, add 0.1 mL of pepsin (100 mg/mL) (Sigma Chemical, St.Louis, Mo.) and digest for 16 hours at 4° Celsius (this removes thepropeptides but leaves the triple helix intact). Centrifuge thesuspension for 20 minutes at 4000×g. Recover supernatant and discard thepellet. Add 2.1 mL of 5M NaCl and 0.5M acetic acid to a final volume of15 mL (final NaCl concentration of 0.7M). Precipitate for approximately16 hours at 4° Celsius. Centrifuge the suspension for 20 minutes at4000×g and discard the supernatant. Dissolve pellet in 0.5mL of 0.5Macetic acid solution. The purity of the collagen should be at least 90%and may be analyzed by standard methods known in the art such asSDS-PAGE, for example.

Applications Using the Conditioned Media Wound Healing Applications

The conditioned media of the invention may be processed to promote woundand bum healing. When tissue is injured, polypeptide growth factors,which exhibit an array of biological activities, are released into thewound to promote healing. Wound healing is a complex process thatinvolves several stages and is capable of sealing breaches to theintegument in a controlled manner to form functionally competent tissue.The process begins with hemostasis followed by an inflammatory phaseinvolving neutrophils and macrophages. The process continues with thedevelopment of granulation tissue andre-epithelialization to close thewound. Subsequently, scar tissue forms and is remodeled over thesucceeding months to an approximation of the original anatomicalstructure. Ideally, scar tissue is minimal so that healthy tissue,functionally competent tissue which histologically and physiologicallyresembles the original normal tissue, may form.

Each stage of the healing process is controlled by cellular interactionsthrough regulatory proteins such as cytokines, growth factors, andinflammatory mediators as well as cell contact mechanisms. For example,inflammatory mediators such as IL-6, IL-8, and G-CSF induce lymphocytedifferentiation and acute phase proteins, as well as neutrophilinfiltration, maturation and activation, processes that are important inthe inflammatory stages of wound healing. Other examples of regulatoryproteins involved in the wound healing process are VEGF that inducesangiogenesis during inflammation and granulation tissue formation, theBMP's which induce bone formation, KGF that activates keratinocytes andTGF-β1 that induces deposition of extracellular matrix. Table 2 (below)lists the concentration of a number of growth factors determined byELISA (enzyme linked immuno assay) to be in Applicants' conditionedmedium which previously supported the growth of the cells grown inDermagraft® tissue culture. It should be understood that the followinglist is not an all inclusive list of factors and is provided solely tofurther characterize the conditioned medium by providing theconcentration of some of the biologically active factors present in themedium of the invention.

TABLE 2 Growth Factor Concentrations in Conditioned Medium as Measuredby ELISA VEGF 3.2 ng/ml G-CSF 2.3 ng/ml IL-8 0.9 ng/ml IL-8 3.2 ng/mlKGF 1.67 ng/ml  TGF-β 0.8 ng/ml

In chronic wounds, the healing process is interrupted at a pointsubsequent to hemostasis and prior to re-epithelialization, and isapparently unable to restart. Most of the inflammation seen in the woundbed is related to infection, but the inflammation gives rise to anenvironment rich in proteases that degrade regulatory proteins and thusinterfere with the wound healing process.

A variety of methods have been utilized to quantify and characterize themajor molecular components secreted by fibroblasts found in thethree-dimensional tissue cultures TransCyteTM and Dermagraft®). Thehuman matrix proteins and glycosaminoglycans (GAGs) present inTransCyteTM and Dermagraft® include, but are not limited to, collagen I,III, fibronectin, tenascin, decorin, versican betaglycan, syndecan aswell as other components (data not shown). These secreted proteins andGAGs serve major structural functions as well as stimulate celldivision, migration, adhesion and signal transduction. The deposition ofglycosaminoglycans (deposition volume is dependent on period of growth)and collagen (deposition volume is not dependent on period of growth) inthe three-dimensional growth systems are illustrated in FIG. 1. Thecomponents have been measured by ELISA, Western blot analysis, immunohistochemistry and PCR. For example, some of the components found inTransCyteTM include collagen I, III, and VII (RNA), fibronectin,tenascin, thrombospondin 2, elastin, proteoglycans, decorin, versican aswell as other components (data not shown). Activity of these componentsin tissue development, healing, and normal function have been welldescribed. Additionally, Applicants describe certain effects of thehuman bioengineered matrix on cell function in vitro. For example,Applicants have noted that cell proliferation is increased by addingbioengineered matrix. To study its effects on cell proliferation, matrixwas physically removed from TransCyte™ and Dermagraft® and added invarying dilutions to monolayer cultures of human fibroblasts andkeratinocytes. The results of increased cell proliferation are shown inFIG. 2.

Further, as detailed in section 6.3, Applicants note the effect of threedimensional conditioned medium on the preparation and composition ofthree-dimensional tissues was examined by measuring the amount ofcollagen secreted into the extracellular matrix of tissues cultured inthe presence of serum-free medium, medium or three dimensionalconditioned medium. The effect of three dimensional conditioned mediumon the preparation and composition of three-dimensional tissues wasexamined by measuring the amount of collagen secreted into theextracellular matrix of tissues cultured in the presence of serum-freemedium, medium or three dimensional conditioned medium. The conditionedmedium of the invention significantly increases collagen deposition oftissue in vitro as shown in FIG. 4. As the present invention containsmany of the regulatory proteins thought to be important in wound healingand which have been shown to be depleted in in vivo models of woundhealing. Furthermore, in some medical conditions, such as diabetes, someof the regulatory proteins needed for wound healing are in short supply.For example, it has been found in a mouse model of non-insulin-dependentdiabetes (e.g., the db/db mouse) that secretion of VEGF and PDGF andexpression of the PDGF receptor are all depressed in wounds compared tothe levels in wounds of normal mice.

Also, the conditioned media provided by the present invention is alsouseful in the treatment of other types of tissue damage, e.g., traumaticor congenital, wherein the repair and/or regeneration of tissue defectsor damage is desired since many of these growth factors are found inApplicants' conditioned cell media, including, for example, fibroblastgrowth factors (FGFs), platelet derived growth factors (PDGFs),epidermal growth factors (EGFs), bone morphogenetic proteins (BMPs) andtransforming growth factors (TGFs); as well as those which modulatevascularization, such as vascular endothelial growth factor (VEGF),keratinocyte growth factor (KGF), and basic FGF; angiogenesis factors,and antiangiogenesis factors. Stress proteins, such as GR 78 and MSP90induce growth factors such as TGF-β. TGF-β, including TGF β-1, TGF β-2,TGF β-3, TGF β-4 and TGF β-5, regulate growth and differentiation andaccelerate wound healing (Noda et al. 1989, Endocrin. 124: 2991-2995;Goey et al. 1989, J. Immunol. 143: 877-880, Mutoe et al. 1987, Science237: 1333-1335). Mitogens, such as PDGF increase the rate of cellularityand granulation in tissue formation (Kohler et al. 1974, Exp. Cell. Res.87: 297-301). As previously mentioned, the cells are preferably human tominimize immunogenicity problems.

Because the conditioned media of the invention contains such an array ofwound healing factors, the conditioned media is advantageously used inthe treatment of wound and bum healing including skin wounds, brokenbones, gastric ulcers, pancreas, liver, kidney, spleen, blood vesselinjuries and other internal wounds. Further, the conditioned media maybe combined with other medicinal ingredients such as antibiotics andanalgesics. Embodiments include formulations of the conditioned mediawith a salve or ointment for topical applications. In fact, theconditioned medium of the invention has been shown to induceproliferation of human fibroblasts and keratinocytes. An increase incell response was noted by cells exposed to the conditioned medium in aslittle as 3 days in vitro (FIG. 3).

Alternatively, the conditioned medium may be combined with a bandage(adhesive or non-adhesive) to promote and/or accelerate wound healing.The conditioned media may be used in any state, i.e., liquid or solid,frozen lyophilized or dried into a powder, as a film for topical woundtreatments and anti-adhesion applications, as an injectable, see PCT WO96/39101, incorporated herein by reference it its entirety.

Alternatively, the conditioned cell medium of the present invention maybe formulated with polymerizable or cross-linking hydrogels as describedin U.S. Pat. Nos. 5,709,854; 5,516,532; 5,654,381; and WO 98/52543, eachof which is incorporated herein by reference in its entirety. Examplesof materials which can be used to form a hydrogel include modifiedalginates. Alginate is a carbohydrate polymer isolated from seaweed,which can be cross-linked to form a hydrogel by exposure to a divalentcation such as calcium, as described, for example in WO 94/25080, thedisclosure of which is incorporated herein by reference. Alginate isionically cross-linked in the presence of divalent cations, in water, atroom temperature, to form a hydrogel matrix. As used herein, the term“modified alginates” refers to chemically modified alginates withmodified hydrogel properties.

Additionally, polysaccharides which gel by exposure to monovalentcations, including bacterial polysaccharides, such as gellan gum, andplant polysaccharides, such as carrageenans, may be cross-linked to forma hydrogel using methods analogous to those available for thecross-linking of alginates described above.

Modified hyaluronic acid derivatives are particularly useful. As usedherein, the term “hyaluronic acids” refers to natural and chemicallymodified hyaluronic acids. Modified hyaluronic acids may be designed andsynthesized with preselected chemical modifications to adjust the rateand degree of cross-linking and biodegradation.

Covalently cross-linkable hydrogel precursors also are useful. Forexample, a water soluble polyamine, such as chitosan, can becross-linked with a water soluble diisothiocyanate, such as polyethyleneglycol diisothiocyanate.

Alternatively, polymers may be utilized which include substituents whichare cross-linked by a radical reaction upon contact with a radicalinitiator. For example, polymers including ethylenically unsaturatedgroups which can be photochemically cross-linked which may be utilized,as disclosed in WO 93/17669, the disclosure of which is incorporatedherein by reference. In this embodiment, water soluble macromers thatinclude at least one water soluble region, a biodegradable region, andat least two free radical-polymerizable regions, are provided. Examplesof these macromers are PEG-oligolactyl-acrylates, wherein the acrylategroups are polymerized using radical initiating systems, such as aneosin dye, or by brief exposure to ultraviolet or visible light.Additionally, water soluble polymers which include cinnamoyl groupswhich may be photochemically cross-linked may be utilized, as disclosedin Matsuda et al., ASAID Trans., 38: 154-157 (1992).

The preferred polymerizable groups are acrylates, diacrylates,oligoacrylates, dimethacrylates, oligomethacrylates, and otherbiologically acceptable photopolymerizable groups. Acrylates are themost preferred active species polymerizable group.

Naturally occurring and synthetic polymers may be modified usingchemical reactions available in the art and described, for example, inMarch, “Advanced Organic Chemistry”, 4th Edition, 1992,Wiley-Interscience Publication, New York.

Polymerization is preferably initiated using photo initiators. Usefulphoto initiators are those which can be used to initiate polymerizationof the macromers without cytotoxicity and within a short time frame,minutes at most and most preferably seconds.

Numerous dyes can be used for photopolymerization. Suitable dyes arewell known to those of skill in the art. Preferred dyes includeerythrosin, phloxime, rose bengal, thonine, camphorquinone, ethyl eosin,eosin, methylene blue, riboflavin, 2,2-dimethyl-2-phenylacetophenone,2-methoxy-2-phenylacetophenone, 2,2-dimethoxy-2-phenyl acetophenone,other acetophenone derivatives, and camphorquinone. Suitable cocatalystsinclude amines such as N-methyl diethanolamine, N, N-dimethylbenzylamine, triethanolamine, trithylamine, dibenzyl amine,N-benzylethanolamine, -isopropyl benzylamine. Triethanolamine is apreferred cocatalyst.

In another embodiment, the conditioned media of the invention, oralternatively particular extracellular matrix proteins elaborated intothe media, are used to provide an excellent substance to coat sutures.The naturally secreted extracellular matrix provides the conditionedmedia with type I and type III collagens, fibronectin, terascin,glycosaminologycans, acid and basic FGF, TGF-α and TGF-β, KGF, versican,decorin and various other secreted human dermal matrix proteins.Similarly, the conditioned cell media of the invention or theextracellular matrix proteins derived from the conditioned media may beused to coat conventional implantation devices, including vascularprosthesis, in surgical approaches to correct defects in thebody-resulting in superior implantation devices. The implants should bemade of biocompatible, inert materials that replace or substitute forthe defective function and made of either non-biodegradable materials orbiodegradable materials. By coating implantation devices with the mediumcontaining these extracellular proteins, the implant invites propercellular attachments resulting in superior tissue at the implantationsite. Thus, sutures, bandages, and implants coated with conditioned cellmedia, or proteins derived from the media, enhance the recruitment ofcells, such as leukocytes and fibroblasts into the injured area andinduce cell proliferation and differentiation resulting in improvedwound healing.

In another embodiment, the conditioned medium may be formulated with apharmaceutically acceptable carrier as a vehicle for internaladministration. Also, the medium may be further processed to concentrateor reduce one or more factor or component contained within the medium,for example, enrichment of a growth factor using immunoaffinitychromatography or, conversely, removal of a less desirable component,for any given application as described herein.

Of course, wounds at specialized tissues may require medium conditionedby that specialized tissue. For example, injuries to neuronal tissuesmay require proteins contained in medium conditioned by neuronal cellcultures. Specific products may be derived, or alternatively, theconditioned medium may be enriched by immunoaffinity chromatography orenhanced expression of a desired protein from the specific medium suchas, for example, NGF. NGF-controlled features include, but are notlimited to, the cholinergic neurotransmitter function(acetylcholinesterase (AChE) and the acetylcholine-synthesizing enzyme(ChAT)), neuronal cell size, and expression of Type II NGF receptors;NGF is secreted into the conditioned medium conditioned by glial andother neuronal cells cultured on a three-dimensional stromal tissue,which can then be used in a composition for nerve healing.

Deficits of endogenous NGF aggravate certain human neurodegenerativedisorders and there is an apparent inability of injured adult CNSneurons to regenerate. Specifically, injury to a nerve is followed bydegeneration of the nerve fibers distal to the injury, the result ofisolation of the axon from the cell body. In the central nervous system,there is no significant growth at the site of injury typically leadingto death of the damaged neuron. NGF plays a crucial role in theregenerative capabilities of adult CNS cholinergic neurons at the cellbody level (e.g., septum), the intervening tissue spaces (e.g., nervebridge) and the reinervation area (e.g., hippocampal formation).Additionally, NGF may be beneficial in improving cognitive defects.Medium conditioned with glial cells for example, can supply exogenousNGF and other nerve growth factors so that new axons can grow out fromthe cut ends of the injured nerve (e.g., develop a growth cone)elongating to the original site of the connection.

Further, injury to the brain and spinal cord is often accompanied by aglial response to the concomitant axonal degeneration, resulting in scartissue. This scar tissue was initially thought to be a physical barrierto nerve growth, however, of greater significance is the presence orabsence of neuronotropic factors in the extra neuronal environment.Astrocytes appear to be capable of synthesizing laminin in response toinjury (laminin can also be found in the conditioned media as discussedin greater detail in Section 5.8.2 relating to extracellular matrixproteins). Collagen and fibronectin, and especially laminin, have beenfound to promote the growth of neurities from cultured neurons orneuronal explants in vitro. These extracellular matrix proteins appearto provide an adhesive substratum which facilitates the forward movementof the growth cone and elongation of the axon. Thus, the presence ofneuronotropic factors and a supportive substratum are required forsuccessful nerve regeneration since regeneration appears to requirethat: the neuronal cell body be capable of mounting the appropriatebiosynthetic response; and the environment surrounding the injury sitebe capable of supporting the elongation and eventual functionalreconnection of the axon. Medium conditioned by nerve cells such asastrocytes and glial cells contains the neuronotropic growth factors andextracellular matrix proteins necessary for nerve regeneration in brainand spinal cord injuries. Thus, in one embodiment, the conditionedmedium is formulated for the treatment of such injuries.

In other embodiments, the treatment of skin, bones, liver, pancreas,cartilage, and other specialized tissues may be treated with mediaconditioned by their respective specialized cell types, preferablycultured in three-dimensions, resulting in a conditioned mediumcontaining characteristic extracellular proteins and other metabolitesof that tissue type useful for treating wounds to that respective tissuetype.

The conditioned cell medium may also be added to devices used inperiodontal surgery in order to promote uniform tissue repair, toprovide biodegradable contact lenses, corneal shields or bone grafts, toprovide surgical space fillers, to promote soft tissue augmentation,particularly in the skin for the purpose of reducing skin wrinkles, andas urinary sphincter augmentation, for the purpose of controllingincontinence.

In another embodiment, the compositions may be lyophilized/freeze-driedand added as a wound filler (e.g., fill holes left from hair plugs forimplantation) or added to existing wound filling compositions toaccelerate wound healing. In another embodiment, the medium isconditioned with genetically engineered cells to increase theconcentration of wound healing proteins in the medium. For example, thecells may be engineered to express gene products such as any of thegrowth factors listed above.

The Repair and Correction of Congenital Anomalies, Acquired Defects andCosmetic Defects

The medium compositions may also be used to repair and correct a varietyof anomalies, both congenital and acquired as well as cosmetic defects,both superficial and invasive. For example, the compositions may beadded in any form and may be used in a hydrogel, injectable, cream,ointment, and may even be added to eye shadow, pancake makeup, compactsor other cosmetics to fortify the skin topically.

In another embodiment, topical or application by any known method suchas injection, oral, etc., of the conditioned medium is made to reverseand/or prevent wrinkles and a number of the deleterious effects inducedby UV light, exposure to a variety of pollutants and normal aging forexample.

Additionally, in another embodiment, the medium of the invention is usedto reduce cell aging and the inhibit the activity of the factors whichcause skin cancer. That the conditioned medium has antioxidant activityis shown in Section 7.1. Again, application to a mammal may be topicalor application by any known method such as injection, oral, etc.Applicants have discovered that a statistically significant (p<0.003)reduction in intracellular oxidation of approximately 50 percent wasnoted in human keratinocytes exposed to Applicants' conditioned medium.

Thus, in addition to inducing epidermal and dermal cell proliferationand collagen secretion in vitro the conditioned medium of the inventionhas strong antioxidant activity (FIG. 5). Also, the factors arerelatively stable and TGF β, VEGF, and collagen content were stableafter 21 days storage at 37° C. at pH 7.4 and 5.5. Solutions stored 2+years at −20° C. maintained stable levels of TGF β1 and VEGF.

This sterile enriched nutrient solution represents a bioengineeredcosmeceutical that is readily available in large volumes and may beuseful as an additive for a variety of skin, cosmetic, and dermatologicproducts to supplement the levels of growth factors and matrix moleculesin human skin, hair, and nails. Products are envisioned to use withAlpha Hydroxy Acids exfoliates to potentially optimize penetration ofthe growth factors and other biomolecules into the skin and withchemical peels to potentially accelerate healing and reduceinflammation.

The conditioned medium may be formulated for eliminating wrinkles, frownlines, scarring and other skin conditions instead of using silicone orother products to do so. The conditioned medium contains growth factorsand inflammatory mediators such as, for example, VEGF, HGF, IL-6, IL-8,G-CSF and TFGβ, (See Table 3, in Section 5.8.1) as well as extracellularmatrix proteins such as type I and type III collagens, fibronectin,tenascin, glycosaminologycans, acid and basic FGF, TGF-α and TGF-β, KGF,versican, decorin betaglycens, syndean and various other secreted humandermal matrix proteins which are useful in repairing physical anomaliesand cosmetic defects. As detailed in section 6.3, Applicants note theeffect of three dimensional conditioned medium on the preparation andcomposition of three-dimensional tissues was examined by measuring theamount of collagen secreted into the extracellular matrix of tissuescultured in the presence of serum-free medium, medium or threedimensional conditioned medium. The conditioned medium of the inventionsignificantly increased collagen deposition of tissue in vitro as shownin FIG. 4. The effect of three dimensional conditioned medium on thepreparation and composition of three-dimensional tissues was examined bymeasuring the amount of collagen secreted into the extracellular matrixof tissues cultured in the presence of serum-free medium, medium orthree dimensional conditioned medium. Of course, the cells used tocondition the medium may be genetically engineered to express improvedconcentrations of such proteins in the medium.

The conditioned media of the invention can be formulated into injectablepreparations. Alternatively, products derived from the conditioned mediacan be formulated. For example, biologically active substances, such asproteins and drugs, can be incorporated in the compositions of thepresent invention for release or controlled release of these activesubstances after injection of the composition. Exemplary biologicallyactive substances can include tissue growth factors, such as TGF-, andthe like which promote healing and tissue repair at the site of theinjection. Methods of product purification include, but are not limitedto gel chromatography using matrices such as SEPHADEX®, ion exchange,metal chelate affinity chromatography, with an insoluble matrix such ascross-linked agarose, HPLC purification, hydrophobic interactionchromatography of the conditioned media. Such techniques are describedin greater detail in Cell & Tissue Culture; Laboratory Procedures,supra; Sanbrook et al., 1989, Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Lab Press, Cold Spring Harbor, N.Y.

In the injectable embodiment, an aqueous suspension is used and theformulation of the aqueous suspension will typically have aphysiological pH (i.e., about pH 6.8 to 7.5). Additionally, a localanesthetic, such as lidocaine, (usually at a concentration of about 0.3%by weight) is usually added to reduce local pain upon injection. Thefinal formulation will also typically contain a fluid lubricant, such asmaltose, which must be tolerated by the body. Exemplary lubricantcomponents include glycerol, glycogen, maltose and the like. Organicpolymer base materials, such as polyethylene glycol and hyaluronic acidas well as non-fibrillar collagen, preferably succinylated collagen, canalso act as lubricants. Such lubricants are generally used to improvethe injectability, intrudability and dispersion of the injectedbiomaterial at the site of injection and to decrease the amount ofspiking by modifying the viscosity of the compositions. The finalformulation is by definition the processed conditioned cell media in apharmaceutically acceptable carrier.

The processed conditioned medium is subsequently placed in a syringe orother injection apparatus for precise placement of the conditionedmedium at the site of the tissue defect. In the case of formulations fordermal augmentation, the term “injectable” means the formulation can bedispensed from syringes having a gauge as low as 25 under normalconditions under normal pressure without substantial spiking Spiking cancause the composition to ooze from the syringe rather than be injectedinto the tissue. For this precise placement, needles as fine as 27 gauge(200μ I.D.) or even 30 gauge (150μ I.D.) are desirable. The maximumparticle size that can be extruded through such needles will be acomplex function of at least the following: particle maximum dimension,particle aspect ratio (length:width), particle rigidity, surfaceroughness of particles and related factors affecting particle:particleadhesion, the viscoelastic properties of the suspending fluid, and therate of flow through the needle. Rigid spherical beads suspended in aNewtonian fluid represent the simplest case, while fibrous or branchedparticles in a viscoelastic fluid are likely to be more complex.

The above described steps in the process for preparing injectablesecreted human conditioned medium are preferably carried out understerile conditions using sterile materials. The processed conditionedmedium in a pharmaceutically acceptable carrier can be injectedintradermally or subcutaneously to augment soft tissue, to repair orcorrect congenital anomalies, acquired defects or cosmetic defects.Examples of such conditions are congenital anomalies as hemifacialmicrosomia, malar and zygomatic hypoplasia, unilateral mammaryhypoplasia, pectus excavatum, pectoralis agenesis (Poland's anomaly) andvelopharyngeal incompetence secondary to cleft palate repair orsubmucous cleft palate (as a retropharyngeal implant); acquired defects(post-traumatic, post-surgical, post-infectious) such as depressedscars, subcutaneous atrophy (e.g., secondary to discoid lupiserythematosus), keratotic lesions, enophthalmos in the unucleated eye(also superior sulcus syndrome), acne pitting of the face, linearscleroderma with subcutaneous atrophy, saddle-nose deformity, Romberg'sdisease and unilateral vocal cord paralysis; and cosmetic defects suchas glabellar frown lines, deep nasolabial creases, circum-oralgeographical wrinkles, sunken cheeks and mammary hypoplasia. Thecompositions of the present invention can also be injected into internaltissues, such as the tissues defining body sphincters to augment suchtissues.

Other tissue types used to condition the media include but are notlimited to bone marrow, skin, epithelial cells, and cartilage, however,it is expressly understood that the three-dimensional culture system canbe used with other types of cells and tissues.

Alternatively, the conditioned cell medium of the present invention maybe formulated with polymerizable or cross-linking hydrogels as describedin the previous section on wound treatment.

Food Additives And Dietary Supplements

The conditioned media may be used as food additives and formulated intodietary supplements. The conditioned media of the invention containsmany useful nutrients including essential amino acids, minerals, andvitamins in an abundance and variety not found in individual foods orgood groups. Applicants are unaware of a more balanced food item (otherthan breast milk) containing such an extensive array of nutrientsalthough such attempts are made in specially formulated, expensiveliquid formulas available for both adults and babies. The conditionedcell media and/or products derived thereof, can be used as aninexpensive source for a balanced nutritional supplement for weight lossor alternatively for enhancing the nutritional content of food,particularly for third world countries. The medium is sterile and isfree from contamination by human pathogens (i.e., aseptic). Theconditioned medium may be concentrated and/or lyophilized and preferablyadministered in capsules or tablets for ingestion. Alternatively, thecompositions may be directly added to adult or baby food to enhancenutritional content. This rich source of nutrients may be processedrelatively inexpensively and can be invaluable to undernourished elderlypeople, and in particular, to children in underdeveloped countries whereincreased mortality due to poor responses to infection have beenassociated with malnutrition.

Additionally, many trace elements found in the conditioned media, suchas iron and magnesium, are critical for mammalian survival andreproduction, and there is concern that marginal trace elementdeficiency may be a public health problem. The intake of variousessential micronutrients has been suggested to decrease infection aswell as cancer risk by modifying specific phases of carcinogenesis.Micronutrients also enhance the functional activities of the immunesystem and its interacting mechanism of T cells and B cells, Mos, and NKcells specifically by enhancing the production of various cytokines tofacilitate their phagocytic and cytotoxic action against invadingpathogens and/or to destroy emerging premalignant cells in various vitalorgans. See, Chandra, R. K. ed. (1988), Nutrition and Immunology.Contemporary Issues in Clinical Nutrition, Alan R. Liss, New York. Thus,there is a need for a relatively inexpensive source of balancednutrients. Ideal food products for enrichment with the conditioned mediaare breads, cereals and other grain products such as pastas, crackers,etc. Also, the medium may be further processed to concentrate or reduceone or more factor or component contained within the medium, forexample, enrichment of a growth factor using immunoaffinitychromatography or, conversely, removal of a less desirable component,for any given application as described in this section.

Animal Feed Supplement

The compositions may be used as a supplemental to animal feed. In oneembodiment, the conditioned medium contains bovine serum that provides asource of protein and other factors that are beneficial for mammals suchas cattle and other ruminant animals, such as cows, deer and the like.The medium is screened for pathogens and is free of bovine pathogens andmycoplasma. The conditioned medium of the invention is preferablyobtained from cows raised in the United States so that the likelihood ofpathogens is markedly diminished.

Cell Culture Medium

The medium compositions may be “re-used” to culture cells, particularlycells that are difficult to culture in vitro. Conventional growth mediumis typically supplemented with many of the factors already present inApplicants' conditioned medium. Further, the conditioned medium alsocontains factors that promote cell attachment and growth such asextracellular matrix proteins described above. Increasing fibronectin orcollagen concentrations may be beneficial for promoting cell attachmentto a scaffold or culture surface. Rather than add these factors to themedium, conditioned medium may be used for culturing cells and preparingthree-dimensional tissue constructs, such as Dermagraft®. Applicantshave demonstrated that the conditioned medium increases cellproliferation of fibroblasts and keratinocytes, see FIG. 3. Cellulardebris or other particulate matter as well as proteases, lactic acid andother components possibly detrimental to cell growth can be removed fromthe medium prior to its reuse as a cell culture medium. It may also bedesirous to use serum in the conditioned cell medium for thisapplication. Serum also contains attachment factors such as fibronectinand serum-spreading factor which promote cell attachment to thesubstrate. Such attachment is required for the growth of some, but notall, cells in vitro. In addition to providing substances necessary forcell growth, serum may also play a role in stabilizing and detoxifyingthe culture environment. For example, serum has a significant bufferingcapacity and contains specific protease inhibitors, such asα1,-antitrypsin and α₂-macro globulin. Serum albumin, present in highlevels in medium containing, for example, 10% serum, may act as anonspecific inhibitor of proteolysis as well as bind fat-solublevitamins and steroid hormones which can be toxic in their free forms.Serum components may also bind and detoxify heavy metals and reactiveorganics which may be present in the medium components.

Pharmaceutical Applications

The conditioned medium of the invention contain a variety of usefulpharmaceutical factors and components such as growth factors, regulatoryfactors, peptide hormones, antibodies, etc., as described throughout thespecification and are therefore useful for a variety of pharmaceuticalapplications. Also, products which may be added include, but are notlimited to, antibiotics, antivirals, antifungals, steroids, analgesics,antitumor drugs, investigational drugs or any compounds which wouldresult in a complimentary or synergistic combination with the factors inthe conditioned media. As previously discussed, the cells are cultured,and the media recovered under aseptic conditions. Additionally, themedia can be tested for pathogens. If sterilization is done, it must bedone in a manner which minimally affects the desired biological activityas described, supra. The medium may be further processed to concentrateor reduce one or more factor or component contained within the medium,for example, enrichment of a growth factor using immunoaffinitychromatography or, conversely, removal of a less desirable component,for any given application as described therein. In a preferredembodiment, formulations are made from medium conditioned by athree-dimensional cell construct. The three-dimensional cultures producea multitude of growth factors and proteins that are secreted into themedium at optimal physiological ratios and concentrations. See forexample, Table 2 in section 5.8.1. The medium, therefore, provides aunique combination of factors and specified ratios that closelyrepresent those found in vivo. Bovine serum is generally not preferredin this application. It may be preferable to remove cellular debris orother particular matter as well as proteases, lactic acid and othercomponents possibly detrimental to cell growth.

The conditioned media may be formulated into pharmaceuticals in the formof tablets, capsules, skin patches, inhalers, eye drops, nose drops, eardrops, suppositories, creams, ointments, injectables, hydrogels and intoany other appropriate formulation known to one of skill in the art. Fororal administration the pharmaceutical compositions may take the formof, for example, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolae); or wetting agents (e.g., sodium lauryl sulphate). Tablets maybe coated using methods well known in the art. Liquid preparations fororal administration may take the form of, for example, solutions, syrupsor suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

The pharmaceutical formulations of the invention may be delivered to apatient via a variety of routes using standard procedures well known tothose of skill in the art. For example, such delivery may besite-specific, oral, nasal, intravenous, subcutaneous, intradermal,transdermal, intramuscular or intraperitoneal administration. Also, theymay be formulated to function as controlled, slow release vehicles.

Therapeutic products contained in the conditioned media include, but arenot limited to, enzymes, hormones, cytokines, antigens, antibodies,clotting factors, and regulatory proteins. Therapeutic proteins include,but are not limited to, inflammatory mediators, angiogenic factors,Factor VIII, Factor IX, erythropoietin (EPO), alpha-lI antitrypsin,calcitonin, glucocerebrosidase, human growth hormone and derivatives,low density lipoprotein (LDL), and apolipoprotein E, IL-2 receptor andits antagonists, insulin, globin, immunoglobulins, catalytic antibodies,the interleukins (ILs), insulin-like growth factors, superoxidedismutase, immune responder modifiers, BMPs (bone morphogenic proteins)parathyroid hormone and interferon, nerve growth factors, tissueplasminogen activators, and colony stimulating factors (CSFs). Ofcourse, the medium may be further processed to concentrate or reduce oneor more factor or component contained within the medium, for example,enrichment of a growth factor using immunoaffinity chromatography or,conversely, removal of a less desirable component, for any givenapplication as described herein.

Assays commonly employed by those of skill in the art may be utilized totest the activity of the particular factor or factors, thereby ensuringthat an acceptable level of biological activity (e.g., a therapeuticallyeffective activity) is retained by the attached molecule or encapsulatedmolecule.

Thus, the condition cell media, and products derived from the media ofthe invention may be used, for example, to provide insulin in thetreatment of diabetes, nerve growth factor for the treatment ofAlzheimer's disease, factor VIII and other clotting factors for thetreatment of hemophilia, dopamine for the treatment of Parkinson'sdisease, enkaphalins via adrenal chromaffin cells for the treatment ofchronic pain, dystrophin for the treatment of muscular dystrophy, andhuman growth hormone for the treatment of abnormal growth.

Doses of such therapeutic protein agents are well known to those ofskill in the art and may be found in pharmaceutical compedia such as thePHYSICIANS DESK REFERENCE, Medical Economics Data Publishers;REMINGTON′S PHARMACEUTICAL SCIENCES, Mack Publishing Co.; GOODMAN &GILMAN, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, McGraw Hill Publ.,THE CHEMOTHERAPY SOURCE BOOK, Williams and Wilkens Publishers.

The therapeutically effective doses of any of the drugs or agentsdescribed above may routinely be determined using techniques well knownto those of skill in the art. A “therapeutically effective” dose refersto that amount of the compound sufficient to result in amelioration ofat least one symptom of the processes and/or diseases being treated.

Toxicity and therapeutic efficacy of the drugs can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD 50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Additionally, the cells and tissues may be genetically engineered toenhance expression of a desired product such as insulin, for example,and/or to express nucleotide sequences and/or moieties which target thegene products listed above e.g. ribozyme, antisense molecules and triplehelices, which may have an inhibitory effect on target gene expressionand/or activity. This might be advantageous when culturing tissues inwhich specialized stromal cells in the medium may play particularstructural/functional roles, e.g., glial cells of neurological tissue,Kupffer cells of liver, etc.

Stimulation of Hair Growth

The medium may be conditioned using, for example, human hair papillacells. Preferably, the medium conditioned by such cells is grown inthree-dimensions. Hair papilla cells are a type of mesenchymal stem cellthat plays a pivotal role in hair formation, growth and restoration(Matsuzaki et al., Wound Repair Regen, 6:524-530 (1998)). Theconditioned medium is preferably concentrated and applied as a topicalformulation. The conditioned media compositions may be formulated fortopical applications using an agent that facilitates penetration of thecompound into the skin, for example, DMSO, and applied as a topicalapplication for stimulating hair growth.

The compositions of the invention promote or restore hair growth whenapplied topically by providing growth factors and other factors thatincrease epithelial cell migration to hair follicles. In addition to thegrowth factors found in the conditioned media, other compounds, such asminoxidil and antibiotics can be used. During hair growth there is areduction in blood supply during catagen (the transitional phase of thehair follicle between growth and resting phases) and telogen (theresting phase). Biologically active molecules derived from theconditioned cell medium can be determined and optimized for use duringthese phases of hair growth using assays known in the art including thestump-tailed macaque model for male-patterned baldness, see for example,Brigham, P.a., A. Cappas, and H. Uno, The Stumptailed Macaque as a Modelfor Androgenetic Alopecia: Effects of Topical Minoxidil Analyzed by Useof the Folliculogram, Clin Dermatol, 1988,6(4): p. 177-87; Diani, A. R.and C. J. Mills, Immunocytochemical Localization of Androgen Receptorsin the Scalp of the Stumptail Macaque Monkey, a Model of AndrogeneticAlopecia, J. Invest Dermatol, 1994,102(4): p. 511-4; Holland, J. M.,Animal Models of Alopecia, Clin Dermatol, 1988,6(4): p. 159-162; Pan, H.J., et al., Evaluation of RU58841 as an Anti-Androgen in Prostate PC3Cells and a Topical Anti-Alopecia Agent in the Bald Scalp of StumptailedMacaques, Endocrine, 1998, 9(1): p. 39-43; Rittmaster, R. S., et al.,The Effects of N, N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17beta-carboxamide, a 5 alpha-reductase Inhibitor and Antiandrogen, on theDevelopment ofBaldness in the Stumptail Macaque, J. Clin EndocrinolMetab, 1987, 65(1): p. 188-93 (each of which is incorporated byreference in its entirety). Additional models include measuringdifferences in hair follicle proliferation from follicles cultured frombald and hairy areas, a newborn rat model as well as a rat model ofalopecia areata, see, Neste, D. V., The Growth of Human hair in NudeMice, Dermatol Clin., 1996, 14(4): p. 609-17; McElwee, K. J., E. M.Spiers, and R. F. Oliver, In Vivo Depletion of CD8+T Cells Restores HairGrowth in the DEBR Model for Alopecia Areata, Br J Dermatol, 1996,135(2): p. 211-7; Hussein, A. M., Protection Against CytosineArabinowide-Induced Alopecia by Minoxidil in a Rat Animal Model, Int JDermatol, 1995, 34(7): p. 470-3; Oliver, R. F., et al., The DEBR RatModel for Alopecia Areata, J Invest Dermatol, 1991, 96(5): p. 978;Michie, H. J., et al., Immunobiological Studies on the Alopecic (DEBER)Rat, Br J Dermatol, 1990, 123(5): p. 557-67 (each of which isincorporated by reference in its entirety).

EXAMPLES Conditioning the Medium

Human dermal fibroblasts were seeded onto the substrate of the apparatusdescribed in the '766 patent and described in detail above in Sections5.3, 5.4 and 5.6. The substrate is within the casing designed tofacilitate three-dimensional tissue growth on its surface and the cellswere cultured in a closed system in cultured in high glucose DMEM (10%BCS supplemented with 2 mM L-glutamine and 50mg/m1 ascorbic acid) at 37°C. in a humidified, 5% CO₂ atmosphere. After 10 days the cell culturewas removed, fresh medium was added. The cells were cultured for anadditional 4 days as described above. The resulting conditioned medium,having been exposed to the cell and tissue culture for four days (days10-14) was then removed from the individual chambers and pooled. Theconditioned medium (approximately 5 to I 0 liters/pool) was dispensedinto 200 ml aliquots and further concentrated 10- to 20-fold using apositive pressure concentration device having a filter with a 10,000 MWcut-off (Amicon, Beverly, Mass.). The resulting 10 to 20 ml ofconcentrated conditioned medium was dispensed into 1 ml aliquots andfrozen at −20° C. for analysis. A 1× concentration of conditioned mediumresults from 10× conditioned medium added to base medium as a 10%(vol/vol) solution. Likewise, a 1× concentration of “medium” or “serumfree medium” results from 10× medium (i.e., base medium) or 10× serumfree medium (base medium without serum) added to base medium as a 10%(vol/vol) solution which are then used as controls.

Proliferating Activity of Three-Dimensional-Conditioned Medium

Exposure of Fibroblasts and Keratinocytes to the Conditioned Medium

The conditioned medium of Section 6.1, was examined for the ability topromote the proliferation of human fibroblasts and keratinocytes. Humanfibroblasts or human basal keratinocytes were seeded into 96 well plates(5,000 cells/well) and cultured in high glucose DMEM (10% BCSsupplemented with 2 mM L-glutamine and 1× antibiotic/antimycotic)supplemented with 1× final concentration of serum-free-medium, medium,or the three-dimensional conditioned medium as described above inSection 6.1. The cultures were maintained at 37° C. in a humidified, 5%CO₂ atmosphere for 3 days.

Cellular Proliferation

Cellular proliferation was measured using a commercially available,fluorescent-based dye assay that measures total nucleic acid content asan estimation of cell proliferation (CyQuant Cell Proliferation AssayKit, Molecular Probes, Eugene, Oreg.). All assays were performedaccording to the manufacturer's instructions. Medium was removed byblotting and the cells were lysed using lysis buffer containing thegreen fluorescent dye, CyQuant GR dye. The dye exhibits strongfluorescence enhancement when bound to cellular nucleic acids and theamount of fluorescence is proportional to the amount of nucleic acidpresent in the sample. Samples were incubated for 5 minutes in theabsence of light and sample fluorescence was determined using amicrotiter plate reader with filters appropriate for ˜480 nm excitationand ˜520 nm emission maxima. The amount of nucleic acid in each samplewas calculated by comparing the amount of observed fluorescence in eachwell against a standard curve, derived using known concentrations ofcalf thymus DNA as a standard.

As shown in FIG. 3, the cells cultured in the medium containing theconditioned medium resulted in increased cellular proliferation of bothfibroblast and keratinocyte cells when compared to the two controls.

Modulation of Collagen Deposition into Tissues by Three-DimensionalConditioned Medium Wound Healing Applications

The effect of three dimensional conditioned medium on the preparationand composition of three-dimensional tissues was examined by measuringthe amount of collagen secreted into the extracellular matrix of tissuescultured in the presence of serum-free medium, medium or threedimensional conditioned medium.

Nylon scaffolds were laser-cut into 11 mm×11 mm squares, washed in 0.5Macetic acid, rinsed extensively in FBS, and seeded with 12F clinicalfibroblasts at passage 8 (˜38,000/cm²). Cultures were grown in 1 ml ofDMEM (10% BCS supplemented with 2 mM L-glutamine and 1×antibiotic/antimycotic) supplemented with 1× final concentration ofserum-free-medium, medium, or the three-dimensional conditioned mediumas described above in Section 6.1 with the addition of 50 mg/mlascorbate at each feeding. Copper sulfate was added to a finalconcentration of 2.5 ng/ml, and high oxygen (40%, about twiceatmospheric) was maintained by regulated gassing of a standardincubator. Cultures (n=3 or greater) were maintained at 37° C. in ahumidified, 5% CO₂ atmosphere for 10 days. A no ascorbate control wasalso included.

Collagen Isolation

Collagen was isolated and purified to near homogeneity fromthree-dimensional tissue cultures grown in the presence of base mediumsupplemented with a 1× final concentration of serum-free medium, mediumor three dimensional conditioned medium described above. The purity ofthe final samples preparations was estimated by subjecting the purifiedcollagen samples to electrophoresis on gradient SDS-polyacrylamide gels,visualizing the separate protein bands using Coomassie blue, andestimating the amount of collagen-specific alpha-, beta- and gamma-bandscompared to total protein (below). Purification methods yielded similarpatterns in all samples.

Samples were rinsed in PBS, then sterile water, followed by 2-6 hours in0.5M acetic acid. The samples were then digested overnight in 1 mg/mlpepsin (Worthington, Inc.) in 0.012N HCI at 4° C. Samples were clarifiedby centrifugation at 13000 rpm at 4° C. Collagen was precipitated 30-60minutes at 4° C. after addition of 5 M NaCl to a final concentration of0.7M. Precipitated collagen was separated by centrifugation at 13000 rpmat 4° C. for 30 to 60 minutes, and was resuspended in 0.012N HCI.

Analysis

Total protein was determined using a commercially available colorimetricassay kit (Pierce, Inc. BCA assay kit) and assays were performedaccording to the manufacturer's instructions. Bovine skin collagens wereused as a standard (InVitrogen, Carlsbad, Calif.; Cohesion Technologies,Inc., Palo Alto, Calif.) for quantifying total protein.

Samples 10 mg) were then subjected to SDS-PAGE analysis withelectrophoresis on 3-8% gradient gels. The samples of isolated collagenswere then heated to 95° C. in reducing sample buffer. Gels were stainedwith Coomassie Blue, destained, and computer-scanned for visualization.

As shown in FIG. 4, a statistically significant (p<0.05) increase incollagen deposition of about 50% was noted for three-dimensionalcultures treated with three dimensional conditioned medium compared toeither control. The activity could not be attributable to the presenceof medium or serum alone.

Such enhanced deposition of collagen in vivo has a number ofapplications, including wound healing, the treatment of wrinkles andcontour lines that appear with increased age as well as being able topromote matrix deposition over bony-prominences susceptible to pressureulcers in paralyzed or bedridden patients.

Antioxidant Activity Exposure of Epidermal Skin Cells to the ConditionedMedium

Antioxidant agents have demonstrated the potential to reverse/preventcell aging and malignancy. The antioxidant activity of thethree-dimensional conditioned medium was measured on human epidermalskin cells. Human basal keratinocytes (˜100,000/well) were cultured inPetri dishes in the presence of MCDB153 medium (KGM, Clonetics, Inc.)supplemented with 1× three dimensional conditioned medium, medium orserum-free medium controls for 3 days at 37° C. in a humidified, 5% CO₂atmosphere. The medium was removed from each sample, the cells wereisolated from the dish by incubating in the presence of trypsin(Sanbrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Lab Press, Cold Spring Harbor, N.Y.), followed bycentrifugation.

FACS Analysis

The isolated cells were incubated in the presence of 1mMdihydrorhodamine-1,2,3 (Molecular Probes, Eugene, Oreg.) at 37° C. for30 minutes and then analyzed by FACS (fluorescence-activated cellsorting) analysis using a Becton-Dickinson (Franklin Lakes, N.J.)FACSCAN apparatus according to the manufacturer's instructions. Theamount of intracellular oxidation of the reduced dihydrorhodamine dye isdirectly proportional to the amount of detectable intracellularfluorescence of the oxidized state of the dye.

Results

Human keratinocytes exposed to Applicants’ conditioned cell mediumexhibited a statistically significant (p<0.0003) 50% reduction inintracellular oxidation compared to the same cells incubated in thepresence of serum-free or serum-containing medium (see FIG. 5). Theobserved difference do not appear to be attributable to serum factors orascorbic acid that are present in the control samples. Therefore,topical administration of medium conditioned by three-dimensionalcultures may be useful as a cosmeceutical useful for treating orreversing the effects of aging cells.

Occlusive Patch Test Assessing the In Vivo Effects of a BioengineeredCosmeceutical Nutrient Solution of Human Skin Experimental Design

Six consenting adult females (30-60 yr) in good health were enrolled.Exclusion criteria included sensitivity to proteins, skin diseases,damaged skin in or near test sites, diabetes, renal, heart orimmunological disorders, use of anti-inflammatory, immuno suppressive,antihistamine or topical drugs or cosmetics and pregnancy. Test articleswere assigned to test sites (2 sites, 3.8 cm2) on the right or leftforearm of each subject according to a rotational scheme to minimizeposition or order bias. Site 1 obtained vehicle control and site 2obtained treatment (i.e., conditioned medium). Occlusion patches were ofa Webril nonwoven cotton pad with either 0.2 ml of vehicle or treatment.Patches were covered and held by a 3M occlusive, plastic, hypoallergenictape. Occlusion patches were positioned daily on the forearms of 3subjects for 5 consecutive, 24-hour periods. The remaining 3 subjectswere patched daily for 12, consecutive 24-hour periods (treatment stillongoing). On the day following the last patch application, a 2-mm biopsyis taken from each site. This protocol was approved by the IRB for theinvestigative organization, the California Skin Research Institute (SanDiego, Ca.), and is in accordance with Title 21 of the CFR, Parts 50 and56.

Evaluations

Gross observations were graded for glazing, peeling, scabbing,fissuring, hyperpigmentation, and hypopigmentation. Irritation wasscored visually using a 5 point scale and graded numerically forerythema, edema, papules and vesicles (>25% patch site), andidentifiable reactions(<25% patch site), i.e., bulla reaction with orwithout weeping, spreading, and induration. The H & E histologicalassessment by a board certified pathologist included parameters forviable epidermal thickness, epidermal hyperplasia (acanthosis), granularcell layer thickness, inflammatory infiltrate, mitotic figures,appearance of collagen and elastic fibers, and vasculature.

Results

No adverse events were induced by the conditioned medium or control inthe 3 subjects receiving a 5-day treatment. Averages of the dailyirritation scores for the nutrient solution (0.3) and the controls (0.2)indicated both sites frequently showed no visible reaction or erythemaor showed slight, confluent or patchy erythema. Histology (trichromecollagen stain) showed healthy tissue for all parameters measured.Hence, the conditioned medium exhibits agreeable affects on human skin.

Modulation of Human Endothelial Cell Behavior

The effects of conditioned medium and human matrix produced bymatrix-bound fibroblasts on angiogenesis and endothelial cell motilitywere determined. Conditioned medium was produced via passage through thethree dimensional fibroblast culture described herein (described inSections 5.3, 5.4 and 5.6) and was either concentrated (10X) orlyophilized. Human extracellular matrix was physically removed from thetissue after production.

Endothelial Cell Tubule Formation Assay

Endothelial cell tubule formation assay with human umbilical veinendothelial cells (HUVEC) was used to assess angiogenesis. Co-culture ofHUVEC with lyophilised conditioned medium caused an increase in tubuleformation when compared to the negative control (pre-conditioned medium)4.9±9.31 mm and 0.00±0.00 mm respectively; concentrated medium increasedtubule formation to 44.10±1.75 mm; and the human extracellular matrixincreased tubule formation to 39.3±5.6 mm

Wounding Assay

A confluent layer of endothelial cells were scratched and the speed ofclosure of the resulting “wound” was measured used to assess cellmotility. The “wounding” assay was measured as speed of closure in mm/h(millimeters/hour). Endothelial cells treated with lyophilised mediumexhibited a speed of 25.59±12.907 mm/h): the concentrated medium a speedof 39.56±15.87 mm/h. Human extracellular matrix showed no increase inspeed when compared to the negative control (pre-conditioned medium)0.00 mm/h and 27.96±10.01 mm/h for human extracellular matrix andnegative control respectively.

Thus, as illustrated by changes in angiogenesis (tubule formation assay)and cell motility (assessed using the “wounding” assay) the conditionedmedium of the invention is able to modulate the behavior of humanendothelial cells.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Various publications are cited herein, the disclosuresof which are incorporated by reference in their entireties.

What is claimed is:
 1. A medium for making a composition suitable fortopical application, the medium made by a process comprising the stepsof: (a) inoculating fetal or embryonic fibroblasts onto a substrate in anutrient medium; (b) culturing the fetal or embryonic fibroblasts in atwo dimensional culture until the cell culture medium comprises at least1.67 ng/mL keratinocyte growth factor; and (c) removing the medium. 2.The medium for making a composition suitable for topical application ofclaim 1, wherein the medium further comprises collagen.
 3. The mediumfor making a composition suitable for topical application of claim 1,wherein the substrate comprises beads.
 4. The medium for making acomposition suitable for topical application of claim 2, wherein duringthe culturing step the fetal or embryonic fibroblasts are subjected tohypoxic stress to thereby increase the secretion of the keratinocytegrowth factor.
 5. The medium for making a composition suitable fortopical application of claim 1, further comprising the step ofconcentrating the medium removed from the cultured cells.
 6. The mediumfor making a composition suitable for topical application of claim 1,wherein the medium removed from the cultured cells is combined with anacceptable carrier.
 7. The medium for making a composition suitable fortopical application of claim 1, wherein the desired level of the growthfactor and the collagen is a concentration sufficient to treat acosmetic defect when applied topically.
 8. A medium for making acomposition suitable for topical application, the medium made by aprocess comprising the steps of: (a) inoculating mesenchymal cells ontoa substrate in a nutrient medium; (b) culturing the mesenchymal cells ina two dimensional culture until the cell culture medium comprises atleast 1.67 ng/mL keratinocyte growth factor; and (c) removing the mediumfrom the cultured cells.
 9. The medium for making a composition suitablefor topical application of claim 8, wherein the medium further comprisescollagen.
 10. The medium for making a composition suitable for topicalapplication of claim 8, wherein the substrate comprises beads.
 11. Themedium for making a composition suitable for topical application ofclaim 8, wherein during the culturing step the fetal or embryonicfibroblasts are subjected to hypoxic stress to thereby increase thesecretion of the keratinocyte growth factor.
 12. The medium for making acomposition suitable for topical application of claim 8, furthercomprising concentrating the medium removed from the cultured cells. 13.The medium for making a composition suitable for topical application ofclaim 8, wherein the desired level of the growth factor and the collagenis a concentration sufficient to treat a cosmetic defect when appliedtopically.
 14. A medium for making a composition suitable for topicalapplication, the medium made by a process comprising the steps of: (a)inoculating fetal or embryonic fibroblasts onto a substrate in anutrient medium; (b) culturing the fetal or embryonic fibroblasts in atwo dimensional culture under hypoxic stress until the cell culturemedium comprises at least 1.67 ng/mL keratinocyte growth factor; and (c)removing the medium from the cultured cells.
 15. The medium for making acomposition suitable for topical application of claim 14, wherein thesubstrate comprises beads.
 16. The medium for making a compositionsuitable for topical application of claim 14, further comprising thestep of concentrating the medium removed from the cultured cells.